Glu311 and Arg337 Stabilize a Closed Active ... - ACS Publications

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Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases V. R. Viviani,*,† A. Simões,†,‡ V. R. Bevilaqua,‡ G. V. M. Gabriel,‡ F. G. C. Arnoldi,§ and T. Hirano∥ †

Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar), Rodovia João Leme dos Santos, km 110, Itinga, Sorocaba, SP, Brazil ‡ Graduate Program of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil § Ribeirão Preto School of Medicine, São Paulo University, Ribeirão Preto, São Paulo, Brazil ∥ Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan S Supporting Information *

ABSTRACT: Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also providing a base to accept excited oxyluciferin phenol proton, and a countercation to shield the negative charge of E311 and to stabilize excited oxyluciferin phenolate, blue-shifting emission spectra in most beetle luciferases.

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remain enigmatic. Several investigations during the past decades have tried to answer these questions by determining the structure and spectroscopic properties of the emitters, and the structural determinants of bioluminescence colors in beetle luciferases. However, the specific interactions between the luciferase active site and the oxyluciferin emitter remain under scrutinity. Essentially, three general mechanisms were proposed to explain bioluminescence color determination by the luciferase active site:8 (I) nonspecific solvent and polarizability effects,8,9 (II) specific interactions of active site residues with excited oxyluciferin,10 and (III) the geometry of the active site affecting

eetle luciferases are unique among luciferases, because they elicit the emission of bioluminescence in a wide range of colors, from green to red, using essentially the same substrates.1,2 Firefly luciferases usually emit in the green-yellow region; however, their spectrum is pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures.3,4 Click beetle and railoradworm luciferases, especially the latter, emit a wider range of colors from green to red, but the spectrum is pH-insensitive from pH 6 to 8.4 However, at higher pH, click beetle luciferases may also undergo broadening and slighter red-shifts.5 Beetle luciferases are currently used in a wide variety of applications as bioanalytical reagents and as reporter genes to investigate gene expression and cell and tissue markers.6,7 Despite the enzymes eliciting different colors, and being so useful as bioanalytical reagents, the mechanisms and structural determinants of bioluminescence colors and of pH sensitivity © 2016 American Chemical Society

Received: March 22, 2016 Revised: June 21, 2016 Published: July 8, 2016 4764

DOI: 10.1021/acs.biochem.6b00260 Biochemistry 2016, 55, 4764−4776

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Biochemistry

firefly and the green-emitting railroadworm luciferases.43,47 However, because most of these active site residues are invariant or conserved among beetle luciferases, only the identity of their side chain is not enough to explain the origin of different bioluminescence colors in beetle luciferases. Recent evidence showed that the main chain amide bonds could be also involved with oxyluciferin phenolate interaction, modulating bioluminescence colors.49 On the other hand, in contrast to firefly luciferases, in the pH-insensitive click beetle and railroadworm luciferases, far fewer single-point mutations were shown to drastically affect the emission spectra.50,51 Several residues affecting bioluminescence colors in all three families of beetle luciferases were found to be clustered in the 223−235 loop, mainly F/Y224, G228, and T/N226,51−54 and the 351−360 loop,53 which lie near the active site and participate in an important network of polar interactions apparently stabilizing the bottom of the luciferin binding site. The salt bridge between residues E311 and R337 is of special interest because these residues are found at the center of this network.30,34,54 Despite the fact mutations R337K/Q in P. pyralis firefly luciferase48 and E311A in Macrolampis firefly luciferase30 were shown to have important effects on bioluminescence spectra, the specific role of these central residues has not yet been fully addressed. Thus, through site-directed mutagenesis, kinetic studies, and spectroscopic studies with luciferin and amino-luciferin analogue and three recombinant beetle luciferases cloned in our laboratories (Macrolampis sp2 firefly, Phrixotrix railroadworm red-emitting, and Pyrearinus termitilluminans click beetle green-emitting), here we investigated the influence of R337 and E311 on bioluminescence color determination and pH sensitivity.

the rotation of thiazinic rings of excited oxyluciferin.11 Most theoretical and experimental studies support both nonspecific solvent and specific acid−base effects (hypotheses I and II). The major problem in determining the mechanism is to identify the putative emitter in bioluminescence, because oxyluciferin has potentially six forms, including tautomers and anionic species.12 Among them, the keto-phenolate form has been credited as the most likely emitter, although the enol-phenolate and enolate-phenolate are also plausible candidates. The tautomerization between a keto red-emitting and an enol yellow-green-emitting form, under the influence of basic residues on the thiazinic ring side of the luciferin binding site, was originally proposed to explain bioluminescence color.10,13 Later studies showed that the keto form can emit a wide range of colors.14 According to such a hypothesis, the luciferase active site could modulate the resonance forms of excited oxyluciferin. More recently, specific base and electrostatic effects around the oxyluciferin 6′-phenol group were thought to play a major role in bioluminescence color determination.15,16 However, recent theoretical and spectroscopic studies raised again the possibility that the enolphenolate could be an emitter in bioluminescence color determination.17−19 Several beetle luciferases have been cloned and sequenced,20−32 mainly from fireflies. The three-dimensional (3D) structure of the North American firefly luciferase Photinus pyralis (Ppy) in the absence of substrates has been determined,33 and then in the presence of bromoform, showing two binding sites, one amphyphilic and another more polar.34 In the less polar site, corresponding to the bottom of the luciferin binding site, residues R218 and E311 were found, whereas in the more polar and external site, residues E311, R337, and E354 were found. Later the 3D structure of the Japanese Luciola cruciata (Lcr) firefly luciferase was determined in the presence of either the luciferyl-adenylate analogue DLSA or with oxyluciferin and AMP,35 showing a closed active site conformation with the former analogue or an open one with the latter products, respectively. The luciferin binding site in firefly luciferases consists mainly of the following residues and segments: R218, the 244HHGF247 motif, the 314SGGAPLS320 loop, and the 340YGLTETTS347 β-hairpin motif.35−37 The structures of Lampyris turkanensis luciferase35 as well as the structure of the P. pyralis luciferase with the C-terminal domain constrained to the rotated conformation were also determined.38 The latter structure confirms that the C-terminus is important for efficient second-half oxidative reaction,39 although studies with the C-terminally truncated firefly luciferase still reveal weak red bioluminescence activity.40 Regions and residues affecting bioluminescence colors in beetle luciferases were identified by chimerization41−43 and random44,45 and site-directed mutagenesis.46−54 Most of the mutations affecting bioluminescence colors in all three families of bioluminescent beetles fall in subdomain B, which contains most of the active site segments. However, in firefly luciferases, several other single-point mutations spread all over the 3D structures and distant from the active site were found to drastically affect bioluminescence colors, usually resulting in red mutants.44,45 The mutation of the invariant/conserved luciferin binding site residues R218, H245, G/A246, and T343 in P. pyralis firefly luciferase was shown to dramatically affect the bioluminescence spectrum.47,48 Of special interest is the invariant R218, which is located near oxyluciferin phenolate, and whose mutation had dramatic effects on the spectra of

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MATERIALS AND METHODS Plasmids and Beetle Luciferase cDNAs. All beetle luciferase cDNAs were previously cloned in our laboratories.23−26 The cDNAs for Phrixotrix hirtus red-emitting luciferase and Py. termitilluminans (Pte) click beetle luciferase were subcloned into the pCold vector (Takara);38 the cDNA of Macrolampis sp2 luciferase was subcloned in the pPro vector.27 Site-Directed Mutagenesis. Site-directed mutagenesis was performed using an Agilent site mutagenesis kit (catalog no. 200518). The plasmids containing the luciferase cDNAs were amplified using Pf u turbo polymerase and two complementary primers containing the desired mutation, using a thermal cycler (1 cycle at 95 °C, 25 cycles at 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 7 min). After amplification, mutated plasmids containing staggered nicks were generated. The products were treated with DpnI to digest nonmutated parental plasmids and used directly to transform Escherichia coli XL1-Blue cells. The following primers and their respective reverse complements were used: Mac-E311Q, TGC ACC AAA TTG CTT CTG GTG GGC G; Mac-E311D, AAT TTG CAC GAC ATT GCT TCT GGT G; Mac-R337E, CCA GGT ATA GAA CAA GGA TAT GGG C; Mac-R337K, CCA GGT ATA AAA CAA GGA TAT GGG C; Mac-R337Q, CCA GGT ATA CAA CAA GGA TAT GGG C; Pte-R334A, CAG GAT AGC GTG TG CTA CG. 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 an OD600 of 0.4 and then induced at 20 °C with 0.4 mM isopropyl β-D-1thiogalactopyranoside for 18 h. Cells were harvested by 4765


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Figure 1. Beetle luciferase primary structure multiple-sequence alignment (top), showing residues E311 and R337 and interacting residues. Topological map (bottom left) of the P. pyralis firefly luciferase structure according to Conti et al., showing important active site stabilizing interactions including E311 and R337. Three-dimensional homology model (bottom right) of Macrolampis sp2 firefly luciferase (91% identical with P. pyralis luciferase) showing the E311 and R337 salt bridge and the network of polar interactions with the loop between residues 223 and 235 (from ref 49).

0.10 M Tris-HCl (pH 8.0), and luciferin at final concentrations between 0.01 and 1 mM. The KM assays for ATP were performed by mixing 5 μL of 80 mM MgSO4 in a solution containing 10 μL of luciferase, 75 μL of 0.10 M Tris-HCl (pH 8.0), and ATP at final concentrations in the range of 0.02−2 mM. Both assays were performed in triplicate. The KM values were calculated using Lineweaver−Burk plots taking the peak of intensity (I0) as a measure of V0. Bioluminescence Spectra. Bioluminescence spectra were recorded in a ATTO LumiSpectra spectroluminometer (Tokyo, Japan) and also, using a Hitachi F4500 spectrofluorometer. For the in vitro bioluminescence, 5.0 μL of luciferases was mixed with 90 μL of 0.10 M Tris-HCl (pH 8.0), 5 μL of 10 mM Dluciferin, and 5 μL of a 40 mM ATP/80 mM MgSO4 mixture. The effect of pH on bioluminescence spectra was analyzed in 0.10 M phosphate buffer (pH 8.0 and 6.0) and 0.10 M TrisHCl (pH 8.0). Homology Modeling. Homology-based models of Ph. hirtus, Py. termitilluminans, Phrixtrix vivianii, and Macrolampis sp2 luciferases were constructed using as a template the threedimensional structure of L. cruciata luciferase in the presence of DLSA [Protein Data Bank (PDB) entry 2D1S] and of oxyluciferin and AMP (PDB entry 2D1R) as previously described,49 Modeler version 9.9 was used to align the sequences (using the align2d function) and to construct 200

centrifugation at 2500g for 15 min and resuspended in extraction buffer consisting of 0.10 M sodium phosphate buffer, 1 mM EDTA, 1 mM DTT, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail (Roche) (pH 8.0), lysed by ultrasonication, and centrifuged at 15000g for 15 min at 4 °C. The N-terminal histidine-tagged Ph. hirtus and Py. termitilluminans luciferases were further purified by agarosenickel affinity chromatography followed by dialysis, according to established procedures.49 The concentrations of purified luciferases were between 0.5 and 1 mg/mL, and the estimated purity, according to sodium dodecyl sulfate−polyacrylamide gel electrophoresis gels, was ∼90%. Measurement of Luciferase Activity. Luciferase bioluminescence intensities were measured using an AB2200 (ATTO, Tokyo, Japan) luminometer. The assays were performed by mixing 5 μL of a 40 mM ATP/80 mM MgSO4 mixture with a solution consisting of 10 μL of luciferase and 85 μL of 0.5 mM luciferin in 0.10 M Tris-HCl (pH 8.0) at 22 °C. All assays were performed in triplicate. Kinetic Measurements and KM Determination. The effect of pH on the activity was assayed in 0.10 M citrate (pH 5−6.0), phosphate (pH 6−8.0), Tris-HCl (pH 7.5−8.5), and CAPS (pH 8.5−10.0) buffers. The KM assays for luciferin were performed by mixing 5 μL of a 40 mM ATP/80 mM MgSO4 mixture in a solution containing 10 μL of luciferase, 75 μL of 4766


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Figure 2. Model of beetle luciferases active sites showing the interactions and distances among E311, R337, and oxyluciferin phenolate: (A) Pyrearinus termitilluminans luciferase, (B) Phrixotrix vivianii green-emitting luciferase, (C) Macrolampis sp2 firefly luciferase, and (D) Phrixotrix hirtus red-emitting luciferase.

3D models of each sequence.55 Visualization and analyses of the best model of each luciferase were performed using PyMol version 1.4.1.56

beetle luciferases displaying different emission spectra and pH sensitivities: Macrolampis sp2 firefly luciferase (569 nm) that is pH-sensitive, Ph. hirtus railroadworm red-emitting luciferase (623 nm), and Py. termitilluminans click beetle green-emitting luciferase (538 nm) that are pH-insensitive. Modeling Studies. Modeling studies with firefly luciferases showed that, besides the salt bridge between E311 and R337 side chains, the E311 carboxyl side chain is hydrogen bonded with the S284 hydroxyl group and N229 carbonyl amide group in the 223−235 loop,52 whereas the R337 guanidine group is hydrogen bonded with G228 and I226 amide carbonyls also in the 223−235 loop; also, its main chain amide is hydrogen bonded with the S314 OH group (Figure 2). Therefore, this constitutes a network of polar interactions among E311, R337, and the 223−235 loop. The amide group of N229 is also at a water-mediated distance from the guanidine group of R218, which was found to play a critical role in bioluminescence colors.40,44 In the closed conformation, E311 is located 6.8 Å from oxyluciferin phenolate, whereas the R337 guanidine group is located at 7.5 Å (Figure 3), which are consistent with a watermediated interaction with the oxyluciferin phenolate. In the open conformation, E311 is located 6.8 Å from oxyluciferin phenolate, whereas the R337 guanidine group is located at 7.3 Å. Table S1 summarizes the distances of critical active site groups and oxyluciferin in the open and closed conformations. Effect of Mutations of E311 in Macrolampis Firefly Luciferase. Previously, we showed that mutant E311A in Macrolampis firefly luciferase produced red light and was pHinsensitive.29 To further investigate the function of this residue in bioluminescence color determination and pH sensitivity, we then introduced the E311Q mutation, in which a similar side chain but lacking the negative charge was replaced, and the E311D mutation, in which the negative charge is preserved but the side chain is slightly shorter. In both cases, the mutant

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RESULTS Rationale behind Site-Directed Mutagenesis. The three-dimensional structure and modeling studies identified two putative arginine residues at the bottom of the luciferin binding site of firefly luciferases that could be involved in oxyluciferin phenolate stabilization and bioluminescence color determination: R218 and R337.43,47 Furthermore, studies with guanidine with Ph. hirtus red-emitting luciferase indicated the lack of a critical arginine in the active site of this unique luciferase.40,57 Site-directed mutagenesis confirmed that mutation of both residues R218 and R337 affects bioluminescence spectra in firefly luciferase.43,47 However, R218 is invariant among beetle luciferases, being present also in Ph. hirtus redemitting luciferase. Additionally, its mutation by Ser had no effect on the bioluminescence spectrum of both Ph. hirtus redemitting and Pyrearinus larval click beetle green-emitting luciferases, indicating that this arginine may not play a universal role in bioluminescence color determination. On the other hand, whereas R337 is invariant in firefly and click beetle luciferases, in Ph. hirtus red-emitting luciferase the respective position 334 is substituted with Leu (Figure 1). In the 3D structure of firefly luciferases, R337 makes a salt bridge with E31134 (Figure 1) that could be important for stabilizing the active site.30,34 The E311A mutation in Macrolampis firefly luciferase produced a red mutant.26 However, the specific roles of E311 and R337 and their salt bridge in bioluminescence color modulation in beetle luciferases have not yet been fully addressed. Thus, by site-directed mutagenesis, kinetic, and modeling studies, we comparatively investigated the influence of E311 and R337 in bioluminescence color in three model 4767


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Figure 3. Bioluminescence spectra of Macrolampis sp2 firefly luciferase forms (A) wild type, (B) E311Q, and (C) E311D at (black line) pH 8.0 and (gray line) pH 6.0.

Figure 4. Bioluminescence spectra of Macrolampis sp2 firefly luciferase R337 forms (A) wild type, (B) R337K, and (C) R337E at (black line) pH 8.0 and (gray line) pH 6.0.

luciferases produced red light (Figure 3) and were pHinsensitive. It is noteworthy that, in the case of E311Q, the KM for luciferin decreased, indicating a slight increase in affinity for luciferin and a large increase in ATP KM (Table 1). We also prepared the E311H mutant that severely impacted the activity and also produced red light (results not shown). Effect of Mutations of R337 in Macrolampis Firefly Luciferase. To investigate whether R337 was indeed involved in a stabilizing interaction with E311 in Macrolampis firefly luciferase, we produced mutant R337K in which the positive charge was kept and mutant R337E in which there was a charge reversal. It is noteworthy that, in both cases, the mutants produced red light (Figure 4). Additionally, the activity of mutant R337E considerably decreased and its KM for luciferin increased (Table 1), whereas the KM for ATP remained almost unaffected, clearly indicating a specific impact on luciferin binding. In contrast, the KM values for mutant R337K for both luciferin and ATP considerably decreased (Table 1), indicating the increasing affinity for both substrates. The results suggest

that a positive charge is required for luciferin binding, probably by stabilizing the E311 negative charge, whereas an additional negative charge could be detrimental for luciferin binding, because it would repel the neighbor E311 charge and also the luciferin phenolate. Influence of R334 Mutations in pH-Insensitive Luciferases. We first analyzed the effect of the R334A mutation in Py. termitilluminans green-emitting luciferase. It is noteworthy that this mutation resulted in a very red-shifted mutant (575 nm) (Figure 5) and also considerably decreased the KM values for luciferin and ATP, indicating an increase in the affinity of these mutants. Thus, we decided to investigate whether the lack of this arginine residue in Ph. hirtus redemitting luciferase could have an effect on bioluminescence color. Indeed, mutation L334R resulted in an 11 nm blueshifted mutant (Figure 6). Although this shift is small, it is significant. Furthermore, although this mutation had little effect on the KM value for luciferin, it decreased the KM value for ATP. 4768


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Figure 5. Effect of mutations of position 334 in pH-insensitive luciferases: (A) L334R in Ph. hirtus luciferase (B) and R334A in Py. termitilluminans luciferase.

Figure 6. Effects of mutation L334R and of guanidine in Ph. hirtus red-emitting luciferase: (A) wild-type luciferase and (B) L334R mutant. The black line shows data for luciferase without guanidine sulfate and the gray line luciferase with 100 mM guanidine sulfate.

Effect of Guanidine. We then tested the effect of guanidine sulfate on the bioluminescence spectra of R337 mutants and Ph. hirtus L334R mutant luciferase to determine whether guanidine blue-shifts their spectra, which occurs in Ph. hirtus wild-type luciferase, mimicking the lost arginine. In Macrolampis firefly luciferase, guanidine promoted a 16 nm blue-shift on the emission spectra of the R337K mutant. However, in Py. termitilluminans R334A mutant luciferase, guanidine did not have any effect. It is noteworthy that guanidine had no effect on the emission spectrum of Ph. hirtus L334R mutant luciferase (Figure 6), in contrast with the results for the wild-type luciferase and all other previously tested mutants that had L334 at this position, providing compelling evidence that R334 may correspond to the missing arginine responsible for the very redshifted emission in the wild-type luciferase. Luciferyl-adenylate Bioluminescence Spectra. The bioluminescence spectra of luciferyl-adenylate with most beetle luciferases match their own spectra with luciferin and ATP (results not shown). Surprisingly, however, for Py. termitilluminans luciferase, the bioluminescence spectrum with luciferyladenylate is 30 nm red-shifted and much broader than that with luciferin and ATP. The only plausible explanation for such unusual difference is that in the luciferase of Pyrearinus luciferyladenylate may bind to a conformation different from that found upon producing luciferyl-adenylate from luciferin and ATP. The bioluminescence spectrum of mutant R334A with LH2AMP is also more red-shifted in relation to the spectrum with luciferin and ATP, indicating again that LH2AMP binds to

a different conformation. In that case, it seems obvious that the R334A mutation is not enough to fully open the active site, such as in firefly luciferases. The mutation would help to polarize the environment, but the enzyme may undergo further conformational changes during catalysis. Thus, this luciferase may display two different conformations for the emissive step: a constrained one that results upon binding luciferin and ATP responsible for green light emission and a more open one upon directly binding LH2AMP, which is responsible for red-shifted emission. It is known that Pyrearinus luciferase displays a quite high KM for luciferin and slower sustained luminescence kinetics, which are indicators of faster release of the product from the active site, in contrast to flashlike kinetics observed for several firefly luciferases, in which product inhibition results in a fast decay. Thus, to release the product faster, the active site must undergo active site opening to facilitate product release after catalysis. We believe that in this luciferase, LH2AMP finds and binds directly to the open conformer, in contrast to enzyme-formed LH2AMP that must be found in a closed conformation. Bioluminescence with 6′-Aminoluciferin. 6′-Aminoluciferin was shown to work as a pH-insensitive bioluminescent probe to investigate the interaction of the 6′-substituent group of oxyluciferin with the surrounding active site environment of beetle luciferases.16 In a previous work, we showed that in the green-yellow-emitting luciferases, the bioluminescence spectrum with 6′-aminoluciferin is red-shifted, whereas in the redemitting ones, it is blue-shifted in relation to the respective 4769


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Figure 7. Bioluminescence spectra of Phrixotrix hirtus and Pyrearinus termitilluminans wild-type luciferases and their 334 mutants with D-luciferin and 6′-aminoluciferin: (A) Phirtus wild type, (B) Phrixotrix hirtus L334R, (C) Pyrearinus termitilluminans wild type, and (D) Pyrearinus termitilluminans R334A. The black line shows data for D-luciferin and the gray line data for 6′-aminoluciferin.

bioluminescence spectra with wild-type firefly luciferin.58 The results indicated that the surrounding environment is apolar in the more blue-shifted luciferases and more polar in the redshifted ones, and that there are two possible binding conformations that explain bioluminescence colors in beetle luciferases: a polar (P) one responsible for red emission and a nonpolar (N) one responsible for green emission. These results are explained on the basis of the difference in the electron donating abilities between the oxido (O−) and amino (NH2) groups of oxyluciferin phenolate anion and aminooxyluciferin, respectively. A similar trend was found with E311 and R337 mutants in Macrolampis firefly luciferase (Figure 7), in which the bioluminescence spectrum with 6′-aminoluciferin was blueshifted for these red mutants. As previously shown, the λmax value of Py. termitilluminans luciferase with D-luciferin is blue-shifted compared to that with 6′-aminoluciferin, indicating that the environment of the excited states of oxyluciferin phenolate and aminooxyluciferin are different from each other. In the case of the R334A mutant, both the bioluminescence spectra with D-luciferin and 6′aminoluciferin are red-shifted in relation to that of the wild-type enzyme, indicating an increase in the polarity of the active site of this mutant. Interestingly, the relationship between the λmax values of D-luciferin and 6′-aminoluciferin is maintained, indicating that the environments of the excited states of oxyluciferin phenolate and aminooxyluciferin are slightly

different from each other. Therefore, the R334A mutation in Py. termitilluminans luciferase makes its active site more polar but provide environments for D-luciferin slightly different from that for 6′-aminoluciferin. Similarly, the λmax value of Ph. hirtus luciferase with Dluciferin is red-shifted compared to that with 6′-aminoluciferin, suggesting that the environments of the excited states of oxyluciferin phenolate and aminooxyluciferin are similar to each other. However, the λmax values of the Ph. hirtus L334R mutant with D-luciferin and 6′-aminoluciferin are similar to each other (Figure 7), indicating that the excited states of oxyluciferin phenolate and aminooxyluciferin experience different environments. Whereas the bioluminescence spectrum of D-luciferin with the L334R mutant is blue-shifted in relation to that of the wild-type luciferase, the bioluminescence spectra of 6′-aminoluciferin with L334R and wild-type enzyme are identical (Figure 7), indicating that the presence of arginine influences specifically excited oxyluciferin but not aminooxyluciferin. Therefore, R334 of Py. termitilluminans luciferase and the L334R mutant of Ph. hirtus luciferase create a specific environment for excited oxyluciferin.

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DISCUSSION Mutation of E311 and R337 residues was shown to universally affect bioluminescence spectra of beetle luciferases, clearly demonstrating their importance. Three hypotheses are 4770


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Figure 8. Bioluminescence spectra of wild-type Macrolampis firefly luciferase and mutant luciferases with 6′-aminoluciferin: (A) wild type, (B) E311D, (C) E311Q, (D) R337E, and (E) R337K. The black line shows the bioluminescence spectrum with D-luciferin and the gray line the bioluminescence spectrum with 6-aminoluciferin.

Table 1. Summary of the Effect of Mutations of Residues E311 and R337 (P. pyralis luciferase sequence numbers) in Beetle Luciferase on Bioluminescence Spectra and Kinetic Properties λmax[half-bandwidth] (nm)b luciferase pH-sensitive Macrolampis sp2 E311Q E311D R337K R337E pH-insensitive Pyrearinus termitilluminans R334A Phrixotrix hirtus L334R

relative activity (%)a

100 mM GuSO4

6′aminoluciferin

KM (μM)c 100 mM guanidinium with 6′aminoluciferin

luciferin

ATP

598 598 590 602 601

20 7.6 19.5 3 127

53 3.5 5.8 3 56

548

560

80

370

570 608[95] 612[95]

589 608 610

15 7 5.8

15 130 21

pH 8.0

pH 6.0

100 31 40 58 162

573[99] 606[67] 605[63] 605[63] 602[93]

610[79] 603[66] 612[60] 608[53] 608[95]

573

100

544[84]

544

0.045 100 88

575[84] 624[78] 613[95]

588

605 611

a

The relative activities were calculated for each mutant in relation to its wild-type luciferase (100%). bThe estimated error in the bioluminescence spectrum peak was ±2.5 nm. cErrors associated with KM values of ±18%. 4771


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Figure 9. Proposed mechanisms of color modulation by Glu311 and R337 in beetle luciferases.

to conformational changes during the emissive step, which could be closed and hydrophobic (N) for green-yellow emission or polar (P) for orange and red emissions. The different magnitude of the red-shifting effect observed between the pH-sensitive and pH-insensitive green-yellow emitting luciferases could be due to the fact that in pHsensitive luciferases the salt bridge between E311 and R337 could be the main interaction stabilizing the closed active site conformation favorable for green light emission, whereas in pHinsensitive green-yellow-emitting luciferases, other interactions, besides the salt bridge between E311 and R337, may contribute to stabilize the closed conformation, turning the active site more rigid. An Arginine Is Essential for Blue-Shifting Emission Spectra. The results described above confirm the importance of the arginine corresponding to R337 in Macrolampis firefly luciferase (R337 in P. pyralis and R339 in L. cruciata) for both pH-sensitive and pH-insensitive green-yellow-emitting luciferases (R334). The mutation of R337 in both pH-sensitive and pH-insensitive green-yellow-emitting luciferases resulted in red or red-shifted mutants, but with different degrees. In pH-insensitive Py. termitilluminans green-emitting luciferase, however, the R334A mutation resulted in a yellow-emitting luciferase that, despite the 27 nm red-shift, still displays the same half-bandwidth [84 nm (Table 1)]. The results are indicative of a solvatochromic effect, rather than a major conformational change, and indicate that this residue may work in concert with other factors to blue-shift the emission spectrum in the wild-type luciferase; otherwise, this single substitution would result in a red mutant such as in firefly luciferases. Similarly, the bioluminescence spectrum of the R334A mutant with 6′-aminoluciferin becomes more redshifted in relation to that of the wild-type luciferase, indicating an increase in polarity. Although a solvatochromic effect upon mutation of Arg to Ala would be in principle expected to blueshift the emission spectrum instead of red-shifting it, because the guanidine moiety of Arg is more polar than the methyl

proposed here to explain the effect of these mutations. (1) These residues act structurally, stabilizing a closed hydrophobic active site conformation favorable for green light emission. (2) These residues act catalytically by being involved in specific interactions with excited oxyluciferin phenolate. (3) Both effects occur. E311 and R337 Stabilize a Closed Green LightEmitting Conformation in Firefly Luciferases. In Macrolampis firefly luciferase, all mutants of E311 and R337 in which the original side chains were replaced with similar side chains lacking the respective charge (E311Q and R337Q), as well as shorter side chains preserving the same charge (E311D and R337K), or side chains with opposite charge (R337E and E311H), resulted in red mutants. These results confirm the importance of the salt bridge between E311 and R337 for stabilizing a closed hydrophobic active site conformation favorable for green light emission (Figure 8). As previously proposed, the firefly luciferase active site displays two possible conformations, one closed and nonpolar (N) one responsible for green-yellow emissions and another open and polar (P) one responsible for red emissions.54,57,58 Quantum yield studies showed that only the green emission is under the influence of pH whereas the red emission is pHinsensitive.59 The results shown here support the previously proposed hypothesis54 that the salt bridge between E311 and R337 at the bottom of the benzothiazolyl side of the luciferin binding site plays a central role in promoting the pH-mediated shift from the closed conformation to the open one polarizing this part of the active site (Figure 9), resulting in red light emission. In pH-sensitive luciferases, the salt bridge between E311 and R337 is stabilized by a set of more labile hydrogen bonds with polar groups of S284 and N229 as well as less polar groups of I226 and G228 in the 223−235 loop,52 which at acidic pH could be disrupted, opening up the benzothiazolyl side of the luciferin binding cavity and polarizing it. In pH-insensitive luciferases, however, the active site displays only a single more rigid conformation that is much less prone 4772


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cavity left near oxyluciferin phenolate. The presence of a cavity near oxyluciferin phenolate in Ph. hirtus luciferase is also suggested by the fact that among beetle luciferases, this luciferase displays the highest affinities for large 6′-substituted amino analogues (results not shown), indicating the presence of larger cavities that may better accommodate these groups. Thus, the specific blue-shifting effect of the guanidinium ion in the wild-type red-emitting luciferase could be caused by a specific interaction with excited oxyluciferin phenolate, or by shielding the E308 carboxylate free negative charge, making the surrounding environment less polar. Altogether, the results shown here clearly indicate that the arginine corresponding to R337 in P. pyralis luciferase (R339 L. cruciata and R334 Py. termitilluminans) plays an essential role in blue-shifting the spectra in most yellow-green-emitting beetle luciferases. Whereas the invariant R218 (P. pyralis) was also shown to be important for green light emission in firefly and P. vivianii green-emitting luciferases, the absence of an effect of its mutation in Py. termitilluminans and Ph. hirtus red-emitting luciferases indicates that this arginine does not play a universal role in bioluminescence color determination. On the other hand, R337 plays a general role in blue-shifting emissions in firefly and click beetle luciferases, whereas its absence in yellowgreen-emitting luciferases of railroadworms (e.g., I334 in P. vivianii luciferase) could be compensated by R215 or by the addition of exogenous guanidinium ion. Similarly, the lack of an effect of R215 in Py. termitilluminans could be compensated by R334. However, a compensating effect seems unlikely, at least in firefly luciferases, because the mutation of both these arginines displays similar effects on bioluminescence spectra, indicating that these arginines play distinct roles. Thus, although both arginines are located close to oxyluciferin phenolate, facing opposite sides, R218 is found in a more buried and hydrophobic environment, making hydrogen bonds with S347 and S250, whereas R337 is found in a slightly more polar environment, making a salt bridge with E311 and hydrogen bonding with the 223−235 and 351−360 loops. Therefore, what is the specific role of R337 in bioluminescence color emission? The kinetic results with P. pyralis48 and Macrolampis sp2 (shown here) firefly luciferases indicate that R337 and positive charges at this position are important for luciferin binding, whereas their loss or charge reversal had an impact on the luciferin affinity and activity. Because at physiological pH luciferin (pKa ∼ 8.7) enters the active site in the neutral phenol form, it is unlikely that the positive charge at this position acts through electrostatic stabilization of phenolate. Rather, positive charges at position 337 may neutralize the neighboring negative charge of the E311 carboxylate, which could be detrimental for luciferin binding, as shown by the decreasing effect on the KM for luciferin upon mutations of E311 that remove the negative charge. Thus, in the ground state, the presence of a neighboring positive charge may help to stabilize E311, providing a neutral environment for luciferin phenol group hydrogen binding. In the singlet excited state, however, the oxyluciferin properties change. Like luciferin photoexcitation, chemiexcitation of oxyluciferin turns the phenol group extremely acidic (pKa < 1.0), releasing a proton to the surroundings. Therefore, a neighboring weak basic group accepting the released proton and a countercation to stabilize the nascent negative charge of excited oxyluciferin phenolate are required in the active site. As previously proposed, the basicity strength and proximity of such base and counteraction may determine the stabilization energy

group of Ala, the replacement of the longer and partially hydrophobic side chain of arginine (with the exception of guanidine group) by a much shorter methyl group may unshield the negative charge of E308, creating a small cavity that could accommodate water, partially polarizing the microenvironment. Therefore, the symmetric band red-shifting effect on the spectrum upon this mutation could be caused by the removal of a specific interaction between R337 and excited oxyluciferin important for blue-shifting the spectra, or by a general solvatochromic effect caused by the shortening of a hydrophobic side chain and removal of a neutralizing positive charge. Because R334 is conserved in other click beetle luciferases that naturally produce green to orange emissions,2,41,59 R334 cannot be considered a natural determinant of bioluminescence colors but clearly plays a critical role in making the emission of these luciferases more blue-shifted. In contrast, in Ph. hirtus red-emitting luciferase, the respective position 334 is substituted by the shorter Leu; thus, we thought that this position could correspond to the previously proposed missing arginine that is rescued by addition of exogenous guanidine. Indeed, the L334R mutation causes an 11 nm blue-shift. What was more remarkable, however, was the fact that the characteristic blue-shifting effect that guanidine causes on the spectrum of the wild-type redemitting luciferase and its mutants43,58 was completely abolished, giving a compelling indication that the L334 site indeed could correspond to the putative missing arginine in the active site of Ph. hirtus red-emitting luciferase. In the wild-type Ph. hirtus luciferase, the emission maximum of D-luciferin (624 nm) is red-shifted compared to that of 6′-aminoluciferin (611 nm), matching the expected difference in the electron-donating ability between the oxido (O−) group and the amino (NH2) group.16 Thus, in the case of the wild-type luciferase, the data suggest that excited oxyluciferin phenolate and the excited aminooxyluciferin experience similar polar environments. On the other hand, the L334R mutant shows similar emission maxima for D-luciferin (613 nm) and for 6′-aminoluciferin (612 nm). These results do not match the expected difference in the electron-donating abilities of the substituents, suggesting that the environments experienced by the excited oxyluciferin phenolate and the excited aminooxyluciferin in L334R mutant are different from each other: less polar for the excited oxyluciferin phenolate and more polar for the excited aminooxyluciferin. Furthermore, the bioluminescence spectrum with 6′-aminoluciferin was identical for both wild-type and mutant luciferases, indicating that the L334R substitution has no effect on 6′-aminoluciferin. Like the effect of the L334R mutation, guanidinium ions exogenously added as a rescue reagent strongly affect the emission of D-luciferin, inducing a blue-shifted emission (608 nm), whereas they do not affect the spectrum with 6′-aminoluciferin: the peak value of the bioluminescence spectrum of aminoluciferin with guanidine (608 nm) is similar to that of the L334R mutant (610 nm). The results indicate that guanidinium ions may display a specific effect on excited oxyluciferin phenolate, making a less polar active site. Guanidine is known for its ability to easily stick to hydrophobic cavities of proteins, displaying both denaturing activity at high concentrations and the chemical rescuing effect of mutated arginines in some proteins at lower concentrations. In the Ph. hirtus red-emitting luciferase, which naturally displays the substitution of the larger arginine with leucine at position 334, guanidine may bind to the corresponding hydrophobic 4773


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Biochemistry of the excited state affecting bioluminescence spectra.16,58 Therefore, if close enough to phenolate, E311 carboxylate and R337 guanidine could provide such base and counteraction, respectively. However, despite the E311 carboxylate and R337 guanidine group being at hydrogen bonding distances between themselves in the closed conformation, which most likely resembles (but is not identical) the conformation during the emissive step, they are located at a water-mediated hydrogen bonding distance from oxyluciferin phenolate (Table S1). Therefore, the water molecule could play an important role as a base under assistance of E311 carboxylate. Under influence of a surrounding hydrophobic environment, a stronger salt bridge would stabilize the closed apolar conformation keeping the ejected proton near excited oxyluciferin phenolate increasing its covalent bond character in the singlet excited state (Figure 9) with a higher energy, and therefore blue-shifting emission. It is possible that the mobility of the single organized water molecule under such circumstances could be restrained, decreasing the orientation polarizability of the environment around oxyluciferin phenolate, blue-shifting the spectra. It is noteworthy that recent spectroscopic studies of oxyluciferin in the presence of a single water molecule in the gas phase may display a hypsochromic effect.18 But we cannot exclude the possibility that during the step that precedes chemiexcitation, the water molecule could be totally squeezed out from the phenolate binding site, increasing the number of specific interactions between E311 and R337 with oxyluciferin phenolate. In the open conformation, however, the distances between these groups increase and relax, increasing the orientation polarizability of the environment. In the singlet excited state, oxyluciferin phenolate becomes more polar; thus, a surrounding polar environment would decrease the energy gap between ground and excited states, resulting in red-shifted emissions, whereas a surrounding apolar environment would increase their energy gap, blue-shifting the emissions. The polarity is not the only factor that might affect the emission spectrum and by itself does not explain the larger shifts from green to red observed in pH-sensitive luciferases at different pHs, or in the pH-insensitive green- and red-emitting luciferases. The cavity size and its rigidity may also contribute to the shape of the spectra: smaller and more compact cavities, reduced degrees of vibrational freedom of the excited molecule, limiting the available space for water molecules and therefore narrowing and blue-shifting the spectra. Indeed, cavity size and hydrophobicity are intimately related; hydrophobic environments are usually associated with smaller and more compact cavities, which promote stronger polar enzyme−substrate (excited product) interactions. In summary, three mechanisms may explain the effect of R337 and its absence on beetle luciferase bioluminescence spectra: (1) stabilization of a salt bridge with E311, its removal resulting in the active site opening and major polarization with the entrance of water, and thus red light emission; (2) establishing a specific (R337) electrostatic interaction with excited oxyluciferin phenolate, or (3) shielding the negative charge of E311. The first hypothesis is best suited for firefly luciferases, in which pH fluctuations trigger the breakdown of the salt bridge resulting in larger active site conformational changes with the loss of specific enzyme-excited oxyluciferin acid−base interactions and polarization of the microenvironment. In this case, the resulting polarization does not exclusively consist of a simple solvatochromic effect, in which a more limited symmetric red-shift of the emission bands is

expected, but of a major change from an aprotic environment to a more protic one in the presence of water, affecting the distribution between green and red emitters. On the other hand, in pH-insensitive green-emitting luciferase, the smaller and symmetric band red-shift effects upon mutation of this arginine are more consistent with a limited solvatochromic effect and possibly with the loss of a specific electrostatic interaction, but not with a major opening and full polarization of the active site in the presence of water. Similarly, in Ph. hirtus red-emitting luciferase, the blue-shifting effects caused by the L334R mutation or by addition of exogenous guanidine are also more consistent with a limited solvatochromic effect or with the gain of a specific electrostatic interaction, which is in agreement with recent spectroscopic results and theoretical simulations that show that the presence of a cation near excited oxyluciferin phenolate may induce hypsochromic shifts in the emission spectrum.60,61 We believe that the explanation for the effect of R337 lies between these two hypotheses, the arginine guanidinium cation shielding the negative charge of E311 and closing the active site, creating a less polar microenvironment, and then acting as a counteraction after excited state proton transfer to E311 carboxylate (or its water-assisted molecule) favoring blue-shifted emissions.

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CONCLUDING REMARKS

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ASSOCIATED CONTENT

Residues R337 and E311 play a central role in bioluminescence color determination in beetle luciferases through both structural and catalytic effects. Their salt bridge stabilizes a closed hydrophobic conformation that promotes stronger acid−base interactions with excited phenol/phenolate, resulting in green and yellow-green light emission in most luciferases. In pHsensitive firefly luciferases, this salt bridge plays a major role in stabilizing the closed nonpolar conformation, its disruption by pH changes opening and polarizing this part of the luciferin binding site, resulting in red light emission. In pH-insensitive green-yellow-emitting luciferases, however, this salt bridge is further stabilized by a more hydrophobic surrounding environment, being less prone to changes in pH. These residues may also provide a water-assisted base (E311) for excited state proton transfer, and a counteraction (R337) that stabilizes the nascent negative charge of phenolate, keeping the ejected proton near excited oxyluciferin phenolate in a high-energy state. Arginine at this position shields the negative charge of E311, turning the microenvironment around excited oxyluciferin phenolate more apolar, thereby blue-shifting the emission spectra in most beetle luciferases, whereas the lack of this arginine in Ph. hirtus red-emitting luciferase is responsible for its very red-shifted bioluminescence.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00260. A table listing distances in angstroms between functional groups of L. cruciata luciferase E313 carboxylate and R339 guanidine groups and oxyluciferin phenolate (PDF) 4774


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(17) Naumov, P., Ozawa, Y., Ohkubo, K., and Fukuzumi, S. (2009) Strucuture and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence. J. Am. Chem. Soc. 131, 11590−11605. (18) Ghose, A., Rebarz, M., Maltsev, O. V., Hintermann, L., Ruckebusch, C., Fron, E., Hofkens, J., Mely, Y., Naumov, P., Sliwa, M., and Didier, P. (2015) Emission properties of oxyluciferin and its derivatives in water: Revealing the nature of the emissive species in firefly bioluminescence. J. Phys. Chem. B 119, 2638−2649. (19) Pinto da Silva, L., and Esteves da Silva, J. C. G. (2015) Chemiexcitation Induced proton transfer: enolate oxyluciferin as the firefly bioluminophore. J. Phys. Chem. B 119, 2140−2148. (20) De Wet, J. R., Wood, K. V., Helinski, D. R., and DeLuca, M. (1985) Cloning of firefly luciferase cDNA and expression of active luciferase in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 82, 7870− 7873. (21) Tatsumi, H., Masuda, T., Kajiyama, N., and Nakano, E. (1989) Luciferase cDNA from Japanese firefly Luciola cruciata: cloning, structure and expression in E. coli. J. Biolumin. Chemilumin. 3, 75−78. (22) Tatsumi, H., Kajiyama, N., and Nakano, E. (1992) Molecular cloning and expression in E. coli of a cDNA enconding luciferase of a firefly Luciola lateralis. Biochim. Biophys. Acta, Gene Struct. Expression 1131, 161−165. (23) Devine, J. H., Kutuzova, G. D., Green, V. A., Ugarova, N. N., and Baldwin, T. O. (1993) Luciferase from the East European firefly Luciola mingrelica: cloning and nucleotide sequence of cDNA, overexpression in E. coli and purification of the enzyme. Biochim. Biophys. Acta, Gene Struct. Expression 1173, 121−132. (24) Ohmiya, Y., Ohba, N., Toh, H., and Tsuji, F. I. (1995) Cloning, expression and sequence analysis of cDNA for the Japanese fireflies, Pyrocoelia miyako and Hotaria parvula. Photochem. Photobiol. 62, 309− 313. (25) Sala-Newby, G. B., Thomson, C. M., and Campbell, A. K. (1996) Sequence and biochemical similarities between the luciferases of the glow-worm Lampyris noctiluca and the firefly, Photinus pyralis. Biochem. J. 313, 761−767. (26) Ye, L., Buck, L. M., Schaeffer, H. J., and Leach, F. R. (1997) Cloning and sequencing of a cDNA for the firefly luciferase from Photuris pennsilvanica. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1339, 39−52. (27) Viviani, V. R., Silva, A. C. R., Perez, G. L. O., Santelli, R. V., Bechara, E. J. H., and Reinach, F. C. (1999) Cloning and molecular characterization of the cDNA for the Brazilian larval Click-beetle Pyrearinus termitilluminans luciferase. Photochem. Photobiol. 70, 254− 260. (28) Viviani, V. R., Bechara, E. J. H., and Ohmiya, Y. (1999) Cloning, sequence analysis and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures. Biochemistry 38, 8271−8279. (29) Viviani, V. R., Arnoldi, F. G., Brochetto-Braga, M., and Ohmiya, Y. (2004) Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 139, 151−156. (30) Viviani, V. R., Oehlmeyer, T. L., Arnoldi, F. G. C., and Brochetto- Braga, M. R. (2005) A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pHsensitivity in firefly luciferases. Photochem. Photobiol. 81, 843−848. (31) Said Alipour, B., Hosseinkhani, S., Nikkhah, M., Naderi-Manesh, H., Chaichi, M. J., and Osaloo, S. K. (2004) Molecular cloning, sequence analysis, and expression of a cDNA encoding the luciferase from the glow-worm Lampyris turkestanicus. Biochem. Biophys. Res. Commun. 325, 215−222. (32) Viviani, V. R., Amaral, D. T., Prado, R. A., and Arnoldi, F. G. C. (2011) A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties. Photoch. Photobiol. Sci. 10, 1879− 1886.

AUTHOR INFORMATION

Corresponding Author

*Department of Physics, Chemistry and Mathematics, Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos, Rod. João Leme dos Santos, Km 110, Sorocaba (SP), Brazil. E-mail: [email protected]. Telephone: (55) 015 3229-7514. Funding

The authors are grateful to FAPESP (2013/09594-0; 2011/ 23961-0) and CNPq (477616/2012-7) for financial support. Notes

The authors declare no competing financial interest.

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