Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence

Article abstract of DOI:10.1021/jacs.5b03820

Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.

Full text of DOI:10.1021/jacs.5b03820

Communication
pubs.acs.org/JACS
Experimental Support for a Single Electron-Transfer Oxidation
Mechanism in Firefly Bioluminescence
,
†
†
†
‡
†
Bruce R. Branchini,* Curran E. Behney, Tara L. Southworth, Danielle M. Fontaine,
Andrew M. Gulick,§ David J. Vinyard, and Gary W. Brudvig
,∥
‡
†
Department of Chemistry, Connecticut College, New London, Connecticut 06320, United States
Hauptman-Woodward Institute, Buffalo, New York 14203, United States
§
∥
Department of Structural Biology, University of Buffalo, Buffalo, New York 14203, United States
‡Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
*
S Supporting Information
Scheme 1. Original Mechanism of Luc Bioluminescence
ABSTRACT: Firefly luciferase produces light by convert-
ing substrate beetle luciferin into the corresponding
adenylate that it subsequently oxidizes to oxyluciferin,
the emitter of bioluminescence. We have confirmed the
generally held notions that the oxidation step is initiated
by formation of a carbanion intermediate and that a
hydroperoxide (anion) is involved. Additionally, structural
evidence is presented that accounts for the delivery of
oxygen to the substrate reaction site. Herein, we report key
convincing spectroscopic evidence of the participation of
superoxide anion in a related chemical model reaction that
supports a single electron-transfer pathway for the critical
oxidative process. This mechanism may be a common
feature of bioluminescence processes in which light is
produced by an enzyme in the absence of cofactors.
5,6
thioesters, the light yielding oxidation of LH -AMP, the
2
second Luc half-reaction, does not require CoA, metal ions, or
other coenzymes. The mechanism of this uncommon cofactor-
independent Luc oxygenase function that distinguishes the
luciferases from the other superfamily members is the focus here.
The generally accepted mechanism of Luc-catalyzed light
production is based on the formation of a key dioxetanone
he familiar yellow-green glow of the firefly lantern is a
beautiful example of bioluminescence. The biochemical
process for the firefly luciferase (Luc) catalyzed conversion of
T
substrates beetle luciferin (LH ) and Mg-ATP into light,
2
18
including the identification of the emitter oxyluciferin
intermediate (Scheme 1), which was inferred from O labeling
7
,8
(
OxyLH ) and the inhibitory side product dehydroluciferyl-
2
results of chemiluminescence model studies. Dioxetanone
intermediates are commonly considered to be sources of
1
AMP (L-AMP), was established ∼60 years ago by McElroy,
White, and others working with the enzyme from Photinus
pyralis. Bioluminescence is the result of two Luc-catalyzed half-
reactions (Scheme 1): (1) the production of luciferyl-adenylate
9
bioluminescence. As proposed by White et al., the oxidative
Luc reaction is initiated when a presumed active site base
abstracts the C4 proton of LH -AMP producing a carbanion
2
(
LH -AMP) and (2) the reaction of LH -AMP with O to
(Scheme 1). The C4 anion, which can be delocalized through the
2
2
2
produce OxyLH in an electronically excited state. According to
adjacent carbonyl, is thought to react with O to form a peroxy
2
2
2
,3
our biochemical and crystallographic studies, the chemistries
take place in two distinct Luc conformations, related by an ∼140°
rotation of the C-terminal domain, that essentially creates 2
distinct active sites. In fact, Luc is a member of the large acyl-CoA
synthetases, non-ribosomal peptide synthetase adenylation
anion intermediate that is rapidly converted into the dioxetanone
as AMP is released. Because it is problematic that the peroxide
1
0
must form from unactivated O
investigated two alternative mechanisms: (1) a radical
in a spin forbidden process, we
2
3
mechanism based on O abstraction of a C4 H atom (Scheme
a) and (2) a single electron-transfer (SET) mechanism
involving superoxide anion ( OO ) formation (Scheme 2b).
Two important features are common to all three of the
oxidative mechanisms: (1) the delivery of O to the C4 carbon of
2
11
4
2
domains, and luciferases (ANL) superfamily of enzymes that
•
−
form the adenylates of carboxylate substrates, e.g., acetate and the
amino acids, that are substrates for subsequent thioester-forming
4
reactions. The domain alternation mechanism that describes the
2
the LH -AMP and (2) the formation of a hydroperoxide (anion)
overall chemical reactions of the ANL superfamily enzymes
provides a fine example of the structure−function theme in
biochemistry. While Luc possesses a CoA binding site and can
2
Received: April 13, 2015
convert LH -AMP and L-AMP into the corresponding CoA
2
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b03820
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 2. Radical and SET Oxidation Mechanisms
Scheme 3. H245C-Catalyzed 4-HO-LH -AMP Formation
2
corresponding sulfenic acid and rationalized these findings,
which support the involvement of a hydroperoxide intermediate
in the oxidation process (Supporting Information, SI), according
to Scheme 3.
2,3
intermediate. In the structure of a cross-linked variant of wild-
type P. Pyralis Luc (PpyWT), the pantetheine tunnel seen in
other ANL enzymes is intact (Figure 1). At the end of this tunnel
Next we determined that the kinetic isotope effect (k /k ) for
H
D
the reaction of PpyWT with 4-deutero-D-LH -AMP was 2.1 ± 0.2
2
(
Table S3). Our result is consistent with the value (2.3)
15
reported by McCapra for the overall bioluminescence reaction
and with the rate-determining step (RDS) of the oxidative
chemistry requiring removal of either a H or a H atom from C4
+
of LH -AMP. While rate-limiting C4 carbanion formation is
2
consistent with the original and SET mechanisms, only the
radical mechanism requires O to participate directly in the RDS
2
(
Schemes 1 and 2). This is because the abstraction of a H atom
by O would be expected to have a relatively high (∼170−210
2
16
kJ/mol) activation energy. With this in mind, we undertook an
experiment in which PpyWT (200 nM) was incubated with LH2-
AMP (100 nM) for 30 s at 22 °C in 100 mM sodium phosphate
buffer, pH 7.8 (NaPB) under anaerobic conditions. During this
time no light emission was observed. However, when the mixture
Figure 1. View down the putative O /pantetheine tunnel. P. pyralis
2
luciferases are shown in the same orientation in the (A) adenylation and
(
B) oxidative conformations. DLSA, a LH -AMP analog, is bound in the
2
(
0.25 mL) was rapidly injected into 0.25 mL of aerated NaPB in a
active site of both structures. The C4 carbon is highlighted in green. The
inset shows the orientation of the DLSA molecule of panel B in ball-and-
stick representation.
reaction tube placed in a luminometer, a rapid burst of light was
emitted characterized by a 90 ms rise time to maximum intensity
(
Figure 2). A control reaction performed by injecting 0.25 mL of
12
is the C4 carbon of DLSA, a stable N-acyl sulfamate analog of
LH -AMP. Luc contains a conserved glycine residue, Gly446,
2
which lines the tunnel. In related enzymes, this glycine forms a
distorted β-sheet interaction with the amide nitrogens of the
pantethene moiety. A G446I mutation in PpyWT specifically
13
impairs the oxidative reaction, suggesting that O approaches
2
the intermediate through this same tunnel. Side chain rotation of
active site residue His245 further expands the tunnel for access to
C4 (Figure 1) of the adenylate. We believe that the initiation of
the Luc half-reaction is coupled to the His245 side chain motion
that provides O access to the C4 carbon of LH -AMP (Figure
2
2
Figure 2. Bioluminescence emission profiles produced by (red)
1
). The proximity of O to His245 in PpyWT was established
2
14
injection of an anaerobic mixture of PpyWT plus LH -AMP into
2
previously by active site-directed photo oxidation studies.
aerated NaPB and (blue) injection of PpyWT in aerated NaPB into a
We initiated this study by making the H245C variant (Table
S1) of PpyWT reasoning that a Luc-generated hydroperoxide
intermediate could oxidize the side chain thiol providing
evidence for the involvement of this peroxy species in the
oxidation process. Modeling studies suggested that the S atom of
the introduced Cys residue could approach the peroxide within a
distance of ∼2.9 Å (Figure S2). The H245C enzyme retained
nearly complete adenylation activity; however, the overall
specific activity and the oxidative half-reaction rate (based on
photon production) were <1% of the PpyWT values (Table S2).
The basis for the lack of bioluminescence activity was the low
solution of LH -AMP in aerated NaPB. Final concentrations of enzyme
and substrate were 100 and 50 nM, respectively.
2
200 nM PpyWT in aerated NaPB into 0.25 mL of a solution of
100 nM LH -AMP in aerated NaPB that had stood at 22 °C for
2
30 s emitted approximately the same amount of light, but at a
much slower rate (400 ms rise time) (Figure 2). These results
were interpreted to indicate that the RDS had occurred prior to
the addition of O , a result that is consistent with the original and
2
SET mechanisms, but not the radical-based process (Schemes 1
and 2). Furthermore, the results suggested that the anaerobic
incubation enabled the buildup of a Luc-stabilized C4
carbanionic intermediate. To test this notion, we performed
(
3%) yield of OxyLH2.
Encouragingly, in addition to obtaining the expected L-AMP
24%) side product, we observed a new major (72%) product 4-
(
hydroxyluciferyl-AMP (HO-LH -AMP) that formed only in the
H245C-catalyzed reaction (Scheme 3). We determined that
Cys245 was selectively and concomitantly oxidized to the
experiments (SI) in which 4-deutero-LH -AMP (3.5 μM) was
incubated with or without PpyWT (7.0 μM) for 30 s at 22 °C in
NaPB under anaerobic conditions. The mixtures were then
2
2
B
DOI: 10.1021/jacs.5b03820
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Journal of the American Chemical Society
Communication
quenched with 10% v/v acetonitrile-0.25 N HCl and analyzed by
ESI/MS. With enzyme present, ∼95% C4 H-D exchange of the
recovered adenylate was observed, while the control reaction
produced <1% exchange. The results of these experiments taken
together are consistent with the Luc-assisted formation of a C4
carbanionic intermediate as required by the original and SET
mechanisms.
We next sought to take advantage of the major difference
between the original and SET oxidation mechanismsthe
involvement of superoxide anion in the latter. We turned to
electron paramagnetic resonance (EPR), a spectroscopic
technique that unambiguously detects unpaired electron species
directly or through the use of spin trapping reagents.
Probably because of the expected short lifetime of enzyme-
generated superoxide anion and/or the inaccessibility of spin
trapping reagents to the active site, we were unable to detect EPR
signals from bioluminescence reactions. So, we turned to the
−
Figure 3. EPR spectra of BMPO-OO in DMSO at 19.5 °C. (a) LH -
2
OMe + sodium phenoxide + BMPO, (b) simulation (see SI), (c) LH2-
OMe + BMPO, (d) sodium phenoxide + BMPO, (e) BMPO only, (f)
LH -OMe + sodium phenoxide, and (g) sodium phenoxide only.
17
well-established chemical model reaction shown in Scheme 4.
2
Scheme 4. Oxidation of LH -OMe
2
Scheme 5. Generation of BMPO-OO−
Under mild alkaline conditions the methyl ester of luciferin
The results reported here provide biochemical data confirming
a peroxy intermediate in Luc bioluminescence and rare
spectroscopic evidence for the involvement of superoxide
anion in a bioluminescence model reaction. Additionally, it is
likely that the pantethine tunnel and His245 expedite superoxide
(
LH -OMe) is oxidized via a hydroperoxide intermediate into a
2
mixture of 4-hydroxyluciferin methyl ester (4-HO-LH -OMe)
2
and dehydroluciferin methyl ester (L-OMe). Presumably
because of the poor leaving group ability of methoxide ion,
OxyLH and light are not produced. The key feature of this
formation by orienting and directing O very close to the reaction
2
2
reaction is that a hydroperoxide is formed from a C4 anion as
required by the original and SET mechanisms. Because we could
use solvent DMSO to obtain relatively high concentrations of
site (Figure 1). We believe that our model results provide a
compelling basis for the Luc-catalyzed SET oxidation mechanism
(Scheme 2b) rather than the spin forbidden process of the
original and generally accepted process (Scheme 1). Luc
bioluminescence provides another example of the “substrate-
LH -OMe (8.5 mM) and 5-tert-butoxycarbonyl 5-methyl-1-
2
pyrroline N-oxide (BMPO) (85 mM), a superior superoxide
18
20
anion spin trapping reagent, we reasoned that if the model
reaction proceeded via a SET mechanism, we could use EPR to
demonstrate the involvement of superoxide anion. This, in turn,
would enable us to use the model chemistry results to support the
Luc catalyzed oxidation mechanism. We began by repeating the
reaction shown in Scheme 4 with a 2.4 mol excess of sodium
phenoxide over methyl ester (SI). Using ESI/MS methods, we
documented a product distribution very similar to the literature
assisted oxygenases” concept in which the organic substrate
drives the oxidation in the absence of cofactors. The SET
mechanism has been proposed for the coelenterazine-obelin
2
1,22
bioluminescence reaction
newly described
and may also be operative in the
2
3,24
earthworm light emission process that likely
proceeds via an acyl adenylate intermediate and in bacterial
bioluminescence. It will be interesting to see if the SET
mechanism of substrate oxidation to form a corresponding
hydroperoxide intermediate is a unifying feature of the diverse
bioluminescence world and if enzyme variants that further
stabilize key carbanion intermediates and superoxide ion can
improve bioluminescence efficiency.
1
7
report. Interestingly, we also detected the hydroperoxide
(
∼4%) confirming the occurrence of this intermediate in the
model reaction. Then we performed EPR studies on the model
reaction along with a series of controls (Figure 3). EPR
spectroscopy using the spin trap BMPO confirmed the
18
ASSOCIATED CONTENT
Supporting Information
Experimental details of H245C mutagenesis and reaction with
presence of superoxide anion. As shown in Figure 3a, the
■
reaction of LH -OMe and sodium phenoxide in the presence of
*
S
2
•
−
BMPO resulted in the formation of the BMPO + OO adduct
Scheme 5). The BMPO-OO EPR spectrum is an overlapping
doublet of triplets” arising from hyperfine interactions from N
−
(
“
(
LH -AMP; protein characterization; LC/MS analyses; substrate
2
14
syntheses; kinetic isotope effects; anaerobic experiments and
product analyses; model oxidation reaction; additional dis-
cussion material; and EPR spectroscopy of model and luciferase
reactions. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/jacs.5b03820.
1
a = 14.1 G) and H (a = 20.8 G). These hyperfine constants
N H
measured in DMSO are similar to values from previous studies of
−
19
BMPO-OO in water. The simulation in Figure 3b assumes a
−
single conformer of BMPO-OO with anisotropic rotational
correlation times. Detailed simulation parameters are provided in
AUTHOR INFORMATION
Corresponding Author
*brbra@conncoll.edu
the SI. Control experiments (Figure 3c−g) show that BMPO-
■
−
OO is formed only when LH -OMe, sodium phenoxide, and
2
BMPO are all present.
C
DOI: 10.1021/jacs.5b03820
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Notes
(24) Dubinnyi, M. A.; Kaskova, Z. M.; Rodionova, N. S.; Baranov, M.
S.; Gorokhovatsky, A. Y.; Kotlobay, A.; Solntsev, K. M.; Tsarkova, A. S.;
Petushkov, V. N.; Yampolsky, I. V. Angew. Chem., Int. Ed. 2015, 54, 7065.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support provided by the
Air Force Office of Scientific Research FA9550-14-1-0100
(
(
B.R.B.), the National Science Foundation MCB-1410390
B.R.B.) and the Hans & Ella McCollum ’21 Vahlteich
Endowment (B.R.B.). The EPR spectroscopy work was
supported by the Department of Energy, Office of Basic Energy
Sciences, Division of Chemical Sciences grant DE-FG02-
05ER15646 (D.J.V. and G.W.B.). We also thank Justin G.
Stroh for performing mass spectrometry and Marc Zimmer for
the molecular modeling studies.
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