(Received for publication, February 18, 1997, and in revised form, May 27, 1997)
From the Department of Chemistry, Connecticut
College, New London, Connecticut 06320 and ¶ Central Research
Division, Pfizer Inc., Groton, Connecticut 06340
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.
Bioluminescence is a captivating process in which living organisms convert chemical energy into light. Beetle bioluminescence, including perhaps 2000 species of fireflies (1, 2), has provided a fertile area of basic and applied research mainly focused on the North American firefly Photinus pyralis. As Equations 1 and 2 indicate, the key firefly luciferase enzyme functions as a monooxygenase, although it does so without the apparent involvement of a metal or cofactor. By some means, amino acid residues are recruited to promote the addition of molecular oxygen to luciferin which is then transformed to an electronically excited state oxyluciferin molecule and CO2, each containing one oxygen atom from molecular oxygen. The light emission process is extremely efficient; nearly a single photon is emitted per reacted luciferin molecule (3).
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(Eq. 1) |
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(Eq. 2) |
The luciferase-catalyzed reactions provide the basis for a wide variety of biochemical assays, many with important clinical applications (4, 5). Moreover, the luciferase gene (6) is widely used as a reporter in studies of gene expression and regulation (7). Additionally, the cloning and sequencing of P. pyralis luciferase and homologues from several beetles (8) have revealed that these enzymes are closely related to a large family of nonbioluminescent proteins (9, 10) that catalyze reactions of ATP with carboxylate substrates to form acyl adenylates.
The recent solving of the crystal structure of luciferase without bound substrates or ligands revealed an apparently unique molecular architecture consisting of two distinct domains (11); however, a detailed description of the luciferase active site remains elusive. Early chemical modification studies have failed to document the involvement of any specific amino acid residues in substrate binding or enzyme catalysis. Many of these studies have been hampered by the high nucleophilic reactivity of several of the luciferase cysteine sulfhydryl groups. However, the results of mutagenesis and other studies (12-14) substantiate the nonessential role of the luciferase thiols. Additional mutational studies (8, 15-17) have identified individual amino acids and regions of the protein, the modification of which alters the color of bioluminescence. No direct structure-function relationship has emerged to account for the color variation of firefly bioluminescence. We have recently used mutational analysis directed at the structural motif common to the superfamily of adenylate-forming enzymes to demonstrate that Ser-198 is directly involved in determining the Km of substrate MgATP, the overall pKa of bioluminescence, and the emission decay rate kinetics.1
In this study, we initially focused on characterizing the firefly
luciferin binding site. We prepared
BPTC,2 a novel
benzophenone-based dehydroluciferin analog (Fig. 1). Dehydroluciferin
is a potent reversible luciferase inhibitor (18) and a
nonbioluminescent substrate for the adenylation reaction (Equation 1).
We intended to use BPTC as a photoaffinity labeling reagent, fully
expecting the benzophenone photophore (19) to be superior to
azide-based reagents that we had previously investigated without
success.3 We report here that BPTC is a
potent photoinactivating reagent for firefly luciferase, but not a
photoaffinity labeling agent. Instead, we will show that BPTC is a
site-specific photooxidizing reagent that has enabled us to identify a
luciferase active site peptide.
The following items were obtained from the
indicated sources: firefly luciferase (crystallized and lyophilized
powder from P. pyralis, EC 1.13.12.7), MgATP
(equine muscle),
N-p-tosyl-L-lysine
chloromethyl ketone-treated chymotrypsin and sequencing grade
trifluoroacetic acid (Sigma); HHGF (Macromolecular Resources).
4-Cyano-benzophenone (20) was prepared in two steps from
4-cyanobenzaldehyde and phenylmagnesium bromide by the procedure of
Wagner and Siebert (21). HOPhHA was synthesized by a literature method
(22).
Hydrogen sulfide was bubbled
for 2 h at room temperature into a stirred solution of
4-cyanobenzophenone (400 mg, 1.9 mmol) in anhydrous pyridine (2.5 ml)
containing a few drops of triethylamine. Nitrogen then was bubbled
through the solution to remove excess H2S, and the mixture
was rotary evaporated leaving a yellow solid (400 mg, 1.7 mmol, 87%).
The crude product was purified by flash chromatography (silica gel,
acetone:hexanes, 30:70, v/v) to a yellow crystalline solid (240 mg, 1.0 mmol, 53%), m.p. 164-167 °C. IR (KBr) 3336 (s), 3137 (s), 1643 (s), 1425 (m), 1309 (m), 1270 (s) cm1; 1H-NMR
(Me2SO-d6, 250 MHz)
7.54-7.61
(m, 3H), 7.67-7.77 (m, 4H), 7.99 (d, 2H), 9.71 (s, 1H), 10.11 (s, 1H);
13C-NMR (Me2SO-d6, 63 MHz)
127.3, 128.7, 129.2, 129.7, 133.0, 136.7, 138.7, 142.8, 195.3, 199.3; high resolution mass spectrometry (fast atom bombardment)
MH+.
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Ethyl
bromopyruvate was added slowly to 4-benzoylphenylthiocarboxamide (510 mg, 2.1 mmol) in absolute ethanol (60 ml), and the solution was
refluxed under N2 for 1.5 h. The mixture was rotary
evaporated yielding a solid that was washed with cold 50% aqueous
ethanol and dried overnight in vacuo at 50 °C. The
product was obtained as a white solid (549 mg, 1.6 mmol, 78%), m.p.
100-102 °C. IR (KBr) 3118 (w), 2981 (w), 1726 (s), 1645 (s), 1603 (m), 1446 (m), 1288 (s) cm1; 1H-NMR
(CDCl3, 250 MHz)
1.42 (t, 3H, J = 7.1 Hz), 4.44 (q, 2H, J = 7.1 Hz), 7.44-7.66 (m, 3 H), 7.77-7.81 (m, 2 H),
7.87 (d, 2H, J = 8.6 Hz), 8.12 (d, 2H, J = 8.6 Hz), 8.21 (s,
1 H); 13C-NMR (CDCl3, 63 MHz)
14.4, 61.7, 126.8, 127.9, 128.4, 130.0, 130.7, 132.8, 136.0, 137.2, 139.1, 148.6, 161.3, 167.4, 195.8; high resolution mass spectrometry (fast atom
bombardment) MH+.
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To a solution
of ethyl 2-(4-benzoylphenyl)thiazole-4-carboxylate (45 mg, 0.13 mmol)
in ethanol (5 ml) was added 10% NaOH (1.7 ml). The yellow mixture was
refluxed for 0.5 h, cooled to room temperature, and acidified to
pH 3 (litmus) with 6 N HCl. A precipitate formed which was
isolated by centrifugation, repeatedly washed with water, and dried
in vacuo at 40 °C. Recrystallization from ethyl acetate
provides a pure product (33 mg, 0.11 mmol, 81%), m.p. 220-222 °C
(with decomposition). IR (KBr) 3300-2400 (br), 1695 (s), 1645 (s)
cm1; UV(NaPB)
max 256 nm (log
= 4.05)
and 312 nm (log
= 4.31); 1H-NMR
(Me2SO-d6, 250 MHz)
7.55-7.77 (m, 5H),
7.86 (d, 2H, J = 8.1 Hz), 8.13 (d, 2H, J = 8.1 Hz), 8.57 (s,
1 H), 13.28 (br s, ~1H); 13C-NMR
(Me2SO-d6, 63 MHz)
126.5, 128.6, 129.6, 129.8, 130.6, 132.9, 135.7, 136.7, 138.3, 148.6, 162.0, 166.1, 195.0;
high resolution mass spectrometry (fast atom bombardment)
MH+.
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Luciferase activity was determined by a
previously described peak height-based light assay method (14).
Luciferase solution concentrations were determined in NaPB by
absorbance measurements at 278 nm, = 45,560 M
1 cm
1 (23). Luciferase
adenylation activity with 0.1 mM luciferin or BPTC as
substrates was assessed by measuring PPi generation at pH
7.8 and 8.6 using the Sigma Pyrophosphate Reagent.
Luciferase (0.5-0.6 mg/ml) in NaPB was mixed with BPTC (0.8-100 µM), and solutions were irradiated with a Rayonet 350 lamp (emitted light, 300-400 nm) at a distance of 7 cm. During photolysis, all samples were kept on ice under a Pyrex glass filter. In the standard photoinactivation protocol, no special precautions were made to remove O2. To exclude O2, NaPB solutions were bubbled with argon for 20 min and, in some cases, sodium dithionite (2.5 mM) was included.
Kinetic ConstantsThe reversible inhibition of luciferase
by BPTC and HOPhHA was assessed with respect to luciferin and MgATP.
For luciferin (6-40 µM), BPTC and HOPhHA levels ranged
from 2.5 to 20 µM and 70 to 700 µM,
respectively. For substrate MgATP (4-325 µM), BPTC and
HOPhHA concentrations were 0.04-13.0 µM and 20-80
µM, respectively. Inhibitory constants
(Ki) were estimated by linear least squares fits of
data from initial velocity measurements performed in the dark. The data
with each luciferase substrate were analyzed by the method of Dixon
(24), and the Ki for MgATP was obtained using the
method of Cornish-Bowden for uncompetitive inhibitors. An independent
estimate of the BPTC binding constant was made from the irreversible
photoinactivation data in Fig. 2. A plot of 1/kobsd
versus 1/[BPTC] was used to determine
Ki and k3 according to the
equation 1/kobsd = Ki/k3[BPTC] + 1/k3 (24).
Proteolysis and Analysis of Luciferase Peptides
BPTC-inactivated luciferase (5% activity) and control
enzyme preparations (irradiated and dark) were digested with
N-p-tosyl-L-lysine
chloromethyl ketone-treated chymotrypsin (protease:protein, 1:20, w/w)
in NaPB for 2 h at 25 °C and 3 h at 37 °C and then quenched by addition of soybean trypsin inhibitor. Chymotryptic peptides were separated by RP-HPLC (14); specific conditions are
detailed in the figure legends. The peak associated with BPTC photoinactivation was collected from control and inhibited luciferase preparations (Fig. 3), concentrated by lyophilization, purified by
rechromatography, and analyzed by LC/ESMS (14) and N-terminal sequencing (Biotechnology Support Facility, University of Kansas Medical Center).
Model Photooxidation Studies
Solutions of peptide HHGF, BPTC, and various additives were irradiated in NaPB at room temperature as described under "Luciferase Photoinactivation Studies." Aliquots were withdrawn and analyzed by RP-HPLC (14). Elution conditions were 40 °C at a flow rate of 1 ml/min, using 0.1% aqueous TFA containing linear gradients of acetonitrile: 10% (v/v) for 5 min; 20% after 20 min; and 75% after 60 min. HHGF concentrations were determined by peak integration. In a separate experiment, the products from irradiation of HHGF (200 µM) in the presence of BPTC (400 µM) were analyzed by LC/ESMS.
BPTC (Fig.
1) was prepared in three steps in 33%
overall yield from 4-cyanobenzophenone. The synthetic route was modeled
after the preparation of dehydroluciferin developed by White et
al. (25). The BPTC structure was confirmed by high resolution mass spectrometry, IR, UV-visible, 1H- and 13C-NMR
spectroscopy (see "Experimental Procedures"). The high molar absorptivity of BPTC solutions in NaPB (max 312 nm, log
= 4.31) enabled photolysis experiments to be carried out with a
Rayonet 350 nm source. Prolonged irradiation of BPTC up to 2 h did
not alter its absorption spectrum. In the dark, using 0.1 mM BPTC, 6.0 ± 1.0 nmol of pyrophosphate/min/mg
luciferase were released at pH 8.6. Thus, BPTC was a substrate for the
luciferase-catalyzed adenylation reaction at pH 8.6 (although not at pH
7.8) as shown in Equation 1.
Irradiation of solutions of native luciferase (8 µM) and BPTC (0.8-100 µM) effected a time- and concentration-dependent inactivation of bioluminescence activity (Fig. 2). Identical results were obtained when recombinant enzyme was irradiated with several concentrations of BPTC. Equimolar and higher concentrations of BPTC rapidly inactivated luciferase. Interestingly, substoichiometric levels of BPTC also could fully inhibit the enzyme. The failure of gel filtration, dialysis, and incubation with dithiothreitol to regenerate enzyme activity provided further evidence for the irreversible nature of the photoinactivation process (data not shown). Additional control studies (Table I) demonstrated that luciferase retained full activity when irradiated in the absence of BPTC or when incubated in the dark with BPTC, affirming that the photoinactivation of luciferase required BPTC.
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Steady-state kinetic analysis (data not shown) of the effect of BPTC (without irradiation) on luciferase flash height generation demonstrated that BPTC was a very good reversible competitive inhibitor (Ki = 3.0 ± 0.3 µM) with respect to substrate luciferin (Km = 15 ± 4.0 µM) (26). The measured Ki value for BPTC is similar to that reported (18) for dehydroluciferin (Ki = 1.0 µM), the structure BPTC was designed to resemble. With respect to substrate MgATP (Km = 110 µM) (27), BPTC was found to be an uncompetitive inhibitor (Ki = 0.55 ± 0.05 µM). The BPTC Ki value was independently estimated to be 7.5 µM from analysis of the irreversible inactivation data presented in Fig. 2 (inset). Attempts to determine whether luciferin or known analogs could protect against BPTC photoinactivation were unsuccessful because these compounds absorb strongly in the irradiated region. To circumvent this problem, we prepared HOPhHA (Fig. 1), the absorption spectrum of which is transparent between 300 and 400 nm. With respect to luciferin and MgATP, HOPhHA was determined to be a reversible competitive inhibitor (Ki = 160 ± 20 µM) and an uncompetitive inhibitor (Ki = 9.0 ± 1.0 µM), respectively. HOPhHA (20 mM) was effective in preventing BPTC photoinactivation of luciferase by ~75% (Table I), indicating that a substantial portion of the enzyme modification is proximal to the luciferin binding site. A protective effect of only ~45% by MgATP (10 mM) was observed in a similar experiment.
Identification of a Photoinactivated Luciferase Active Site PeptideRP-HPLC was used to separate proteolytic fragments generated from control and photoinactivated luciferase digested with chymotrypsin, trypsin, or endoproteinase Glu-C. Only chymotrypsin digests presented a pattern of peptides with any differences between control and inactivated samples, the sole difference being a ~75% decrease in the area of the peak indicated by an asterisk in Fig. 3. The decrease in area of this peak was directly correlated with the loss of enzyme activity (data not shown). However, no new peaks appeared in the digest of inactivated protein.
The peaks designated in Fig. 3 were further purified by rechromatography (Fig. 3, inset). By Edman sequencing and LC/ESMS, both peaks were found to contain two coeluting peptides, HHGF (MH+ = 497.4) and VRGPM (MH+ = 559.4), corresponding to regions 244-247 and 392-396 in the deduced luciferase sequence (6). The mole percentage of HHGF:VRGPM in the peak from the control sample was estimated from the sequencing data to be 78:22. In the photoinactivated sample, the HHGF content decreased ~70%, while the amount of VRGPM remained unchanged. Therefore, the loss of peptide 244HHGF247 was correlated to the BPTC-mediated photoinactivation process.
Model Photooxidation Studies with HHGFThe absence of any
evidence for BPTC-peptide adduct formation suggested that no true
photoaffinity labeling had occurred during the photoinactivation
process. Instead, it seemed likely that photooxidation of one or both
of the imidazole side chains of 244HHGF247 may be a major cause of
luciferase inactivation. A model study was undertaken in which
synthetic HHGF (200 µM) was irradiated with BPTC (400 µM). As evidenced by the analysis of the reaction
products by LC/ESMS (Fig. 4), BPTC did
catalyze the photooxidation of HHGF. Moreover, the formation of the
putative products (Fig. 4, inset), assigned from the mass
data and by analogy to reported studies (28, 29) on the dye-sensitized
photooxidation of histidine, suggested that BPTC irradiation generates
singlet oxygen. Additional model studies (Table
II) supported the involvement of singlet
oxygen: (i) sodium azide, a scavenger of singlet oxygen prevented
degradation of HHGF; (ii) the use of buffers prepared in deuterium
oxide, which prolongs the lifetime of singlet oxygen, accelerated HHGF
photooxidation 3-fold; (iii) superoxide dismutase and mannitol,
reagents that destroy superoxide and hydroperoxide radicals,
respectively, had very little or no effect; and (iv) reduction of
soluble oxygen by argon bubbling slowed the rate of HHGF
photooxidation, while sodium dithionite, which removes soluble oxygen
(30), nearly halted the process. None of these reagents affected HHGF
photooxidation in the absence of BPTC.
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The results of luciferase photoinactivation experiments in which soluble oxygen was reduced by argon bubbling and sodium dithionite indicated decreased or strongly suppressed enzyme inhibition (Table I). A control trial showed that sodium dithionite had no effect on BPTC stability. Therefore, unlike photoaffinity labeling experiments that are usually retarded by oxygen, BPTC-mediated photoinactivation required it. Since substoichiometric concentrations of BPTC completely inactivated luciferase (Fig. 2) and since BPTC was not consumed in the process (Fig. 3), the benzophenone derivative must function as a photooxidation catalyst. Although sodium azide only modestly protected luciferase against BPTC photooxidation, evidence supporting the involvement of singlet oxygen included (Table I): (i) a 1.7-fold decrease in the inhibition half-life measured in deuterium oxide buffers; and (ii) the failure of superoxide dismutase or mannitol to alter the inactivation rate.
Since Cys, Met, Tyr, Trp, and His are especially susceptible to photooxidation (31), we compared the content of these amino acids in irradiated control and BPTC-inactivated luciferase (Table III). A loss of His consistent with the determined level of 244HHGF247 photooxidation and a very minor decrease in Cys were the only differences noted (including all other amino acids not shown in Table III).
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Molecular modeling was used to design BPTC, a dehydroluciferin analog containing a benzophenone moiety (Fig. 1). BPTC very effectively inactivated luciferase irreversibly by a process requiring irradiation (Fig. 2). Furthermore, luciferase modification occurred at or near the luciferin binding site as: (i) BPTC was found to be a substrate for the adenylation reaction shown in Equation 1; (ii) the aryl ketone derivative was an effective reversible competitive inhibitor (Ki = 3.0 ± 0.3 µM) with respect to luciferin; and (iii) the BPTC-promoted inactivation process was effectively retarded by the luciferin analog HOPhHA (Table I). BPTC also was shown to be an uncompetitive inhibitor (Ki = 0.55 ± 0.05 µM) with respect to another luciferase substrate, MgATP. Since MgATP provided modest protection against photoinactivation, a portion of the luciferase inactivation may occur at a second site, possibly an allosteric binding site for ATP (32). Using proteolysis, RP-HPLC, LC/ESMS, and Edman sequencing (Fig. 3), we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. However, since complete enzyme inhibition was accompanied by the loss of only ~70-75% of the peptide, the BPTC-mediated photoinactivation process must occur at more than one site, possibly a cysteine residue. While HHGF was identified as the major site of luciferase modification by comparison to controls, no modified peptides were found. As suggested by model studies (Fig. 4), several modified HHGF peptides probably were formed, but no single one was produced at a measurable level.
Nature of the Photoinactivation ProcessThe modification of
luciferase at 244HHGF247 could not, as expected for a benzophenone
derivative, be associated with photoaffinity labeling because: (i) the
photoinactivation process required oxygen; (ii) substoichiometric
amounts of BPTC fully inactivated luciferase; (iii) no BPTC-HHGF adduct
was detected; and (iv) BPTC was not consumed during the
photoinactivation process. Instead, BPTC is a catalyst for luciferase
photooxidation. There are many examples of substituted aryl ketones
functioning as triplet sensitizers (35). In the BPTC structure, the
extended conjugation provided by the para-substituted
thiazole ring must shift the key electronic transition from
n-* to
-
*, thereby producing a molecule much less
reactive to H abstraction required for photoaffinity labeling (19). The
extended conjugation in the BPTC structure also may make it a more
efficient triplet sensitizer.
Although it is difficult to distinguish between type II (singlet oxygen) and type I (electron transfer between an excited triplet state and a substrate molecule) photodynamic processes, we propose that BPTC generates singlet oxygen at the luciferase active site in very close proximity to the peptide 244HHGF247. The results of model studies undertaken with synthetic HHGF (Table II; Fig. 4) strongly support our contention that BPTC is a sensitizer for singlet oxygen production. Likewise, evidence consistent with BPTC-promoted singlet oxygen inactivation of luciferase was compiled (Table I). However, two significant differences were noted between the enzyme and model studies: (i) azide ion was much less effective in protecting the protein; and (ii) the enzyme inactivation process was ~150 times faster than the photooxidation of the model peptide. Previously, Seliger and co-workers reported (33) the inactivation of luciferase with singlet oxygen generated by irradiating an immobilized Rose Bengal catalyst. They determined that luciferase was ~10 times more sensitive to singlet oxygen than histidine. The even greater sensitivity of luciferase to BPTC-photooxidation than the actual peptide being modified very likely reflects the close proximity of both oxygen and BPTC to the 244HHGF247 region. The failure of sodium azide to retard luciferase photooxidation in our study presumably reflects the inability of the ion to reach the enzyme site of singlet oxygen generation.
Possible Functional Role of 244HHGF247To explain the greater
sensitivity of luciferase to singlet oxygen, Seliger proposed (33) that
a histidine residue was located at a putative enzyme binding site for
molecular oxygen. Mutagenesis studies support a similar proposal for
the importance of a histidine residue in oxygen binding in the
bioluminescent photoprotein aequorin (34). Possibly, in the firefly
protein, the region 244HHGF247 is involved in binding oxygen for
subsequent addition to the luciferyl adenylate (Equation 2). BPTC
binding near this site would be expected to make His-244 and/or His-245
the most susceptible residue(s) to photooxidation as we have observed.
Alternatively, the tetrapeptide may be involved in luciferin binding.
Interestingly, His-245 is conserved among all of the 13 known firefly
luciferase sequences (8). Preliminary results of ongoing mutagenesis
studies support an important functional role for His-245 since the
mutant proteins H245Q and H245A show altered light emission kinetics
and ~200- and ~400-fold lower relative activities, respectively,
compared with wild-type luciferase. Furthermore, 244HHGF247 is located in the 226-250 region of the luciferase primary sequence in which minor changes produce dramatic shifts in the color of bioluminescence (8, 16). This region is part of the -sheet B subdomain, which is
located in the large N-terminal domain of the luciferase structure
(11). It appears that the tetrapeptide is located near an internal
cavity containing ordered water molecules. The cavity is in a surface
groove close to several invariant residues that comprise the putative
luciferase active site (11). When the x-ray coordinates become
available, it will be possible to better determine the location of the
244HHGF247 in the luciferase three-dimensional structure.
We have described here an uncommon example
of active site-directed photooxidation of an enzyme by singlet oxygen.
At least one other example of site-directed photooxidation has been
reported (36), although the active species responsible for oxidation of
a Cys residue in -3-ketosteroid isomerase was not determined. The
benzophenone group, so useful in photoaffinity labeling studies (19),
has been used here to efficiently catalyze the photooxidation of a
histidine-containing firefly luciferase peptide. Potentially, BPTC and other substituted benzophenones with extended conjugation may
prove generally useful as site-directed photooxidation reagents. We are
currently investigating this possibility and extending the ongoing
mutation studies to better determine the functional role of 244HHGF247
in firefly bioluminescence.
We thank Jeff Kiplinger and Richard Ware for providing the mass spectral analyses of the synthetic compounds and Evelyn Bamford for technical assistance.
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