β-Deuterium Isotope Effects on Firefly Luciferase Bioluminescence

Article abstract of DOI:10.1002/open.201700136

A 5,5-d₂-luciferin was prepared to measure isotope effects on reactions of two intermediates in firefly bioluminescence: emission by oxyluciferin and elimination of a putative luciferyl adenylate hydroperoxide to dehydroluciferin. A negligible isotope effect on bioluminescence provides further support for the belief that the emitting species is the keto-phenolate of oxyluciferin and rules out its excited-state tautomerization, one potential contribution to a bioluminescence quantum yield less than unity. A small isotope effect on dehydroluciferin formation supports a single-electron-transfer mechanism for reaction of the luciferyl adenylate enolate with oxygen to form the hydroperoxide or dehydroluciferin. Partitioning between the dioxetanone intermediate (en route to oxyluciferin) and dehydroluciferin is determined, not by the fate of the hydroperoxide, but by that of the radical formed from luciferyl adenylate, and the kinetic isotope effect (KIE) reflects H-atom abstraction by superoxide.

Full text of DOI:10.1002/open.201700136

DOI: 10.1002/open.201700136
b-Deuterium Isotope Effects on Firefly Luciferase
Bioluminescence
Michael C. Pirrung,*[a, b, c] Allyson Dorsey,[a, b] Natalie De Howitt,[a, b] and Jiayu Liao[b]
A 5,5-d2-luciferin was prepared to measure isotope effects on
reactions of two intermediates in firefly bioluminescence: emis-
sion by oxyluciferin and elimination of a putative luciferyl ade-
nylate hydroperoxide to dehydroluciferin. A negligible isotope
effect on bioluminescence provides further support for the
belief that the emitting species is the keto-phenolate of oxylu-
ciferin and rules out its excited-state tautomerization, one po-
tential contribution to a bioluminescence quantum yield less
than unity. A small isotope effect on dehydroluciferin forma-
tion supports a single-electron-transfer mechanism for reaction
of the luciferyl adenylate enolate with oxygen to form the hy-
droperoxide or dehydroluciferin. Partitioning between the di-
oxetanone intermediate (en route to oxyluciferin) and dehy-
droluciferin is determined, not by the fate of the hydroperox-
ide, but by that of the radical formed from luciferyl adenylate,
and the kinetic isotope effect (KIE) reflects H-atom abstraction
by superoxide.
Scheme 1. Accepted pathway of firefly bioluminescence.
The reaction pathway of firefly bioluminescence is well estab-
lished (Scheme 1).[1] The stoichiometric substrate luciferin (1) is
converted to acyl adenylate (2) that reacts with oxygen to
form presumably the hydroperoxy-adenylate (3),[2,3] an inter-
mediate that has never been directly observed. Two paths can
be envisioned for 3. The first involves conversion via the dioxe-
tanone to carbon dioxide and the excited state of oxyluciferin
(4), which emits. A side reaction diverts some of 3 to hydrogen
peroxide plus dehydroluciferin adenylate (5), which hydrolyzes
to dehydroluciferin (7). Past studies have determined a 6/7
ratio of 4:1.[4]
despite a large number of possible ionic states and tautomeric
forms, the emitting species is the keto-phenolate shown.[5] For
many years, luciferase had been considered to be highly effi-
cient, but careful modern measurements have shown instead a
quantum yield (F) of 0.42.[6] The basis of this quantum yield of
less than unity is not understood, but possibilities for non-radi-
ative loss of the chemically generated excited state 4 include
internal conversion or a reaction such as excited-state ioniza-
tion or tautomerization.[7] The oxyluciferin product 6 had been
believed to be highly unstable, but recent work showed it
simply requires careful handling.[8] Isotopically labeled luciferins
have been used to show the expected primary a-deuterium ki-
netic isotope effect of approximately 2 on formation of the
enolate of 2 for oxygenation.[9]
Many mechanistic details of this process have been worked
out previously. Deep investigations of 4 strongly suggest that,
[a] Prof. M. C. Pirrung, A. Dorsey, N. D. Howitt
Department of Chemistry, University of California
Riverside, CA 92521 (USA)
We became interested in the b-deuterium isotope effect (b-
related to the reaction site at C4 of the thiazoline ring) on the
firefly luciferase reaction, because of its potential to yield in-
sight into multiple features of this classical mechanism. Once
the b-deuterated luciferin enters into this reaction sequence, it
can exert primary isotope effects on two consecutive mecha-
nistic steps. It could also show a b-secondary isotope effect on
the formation of 3, but its magnitude would be small com-
pared to the two primary isotope effects. They are worthy of
study, because they could impact bioluminescent efficiency
and provide a deeper understanding of mechanism.
Fax: 951-827-2749
E-mail: Michael.pirrung@ucr.edu
[b] Prof. M. C. Pirrung, A. Dorsey, N. D. Howitt, Prof. J. Liao
Department of Bioengineering, University of California
Riverside, CA 92521 (USA)
[c] Prof. M. C. Pirrung
Department of Pharmaceutical Sciences, University of California
Irvine, CA 92697 (USA)
Supporting Information for this article can be found under:
https://doi.org/10.1002/open.201700136.
ꢀ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
For example, the strong similarity between the biolumines-
cence spectrum with the native substrate luciferin and a syn-
thetic substrate analog, 5,5-dimethylluciferin, provides experi-
mental evidence that the keto form is the only emitter.[10] As
5,5-dimethylluciferin gives an oxyluciferin excited state that
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cannot tautomerize, there is no possibility of an enol contribu-
ting to its emission. A weakness of that study is the necessity
to pre-synthesize the adenylate of 5,5-dimethylluciferin, be-
cause its adenylate cannot be generated by luciferase. Howev-
er, firefly luciferase accepts the adenylate as a substrate. Di-
deuteration of luciferin in lieu of dimethylation should impede
tautomerization in d2-4, but, unlike 5,5-dimethylluciferin, both
half-reactions of bioluminescence can be studied with d2-1. A
change in bioluminescence with this dideuterated substrate
would signal something other than the keto form being in-
volved in the photochemistry of 4. Kinetic isotope effects on
simple ketone enolizations are 4–6,[11] and enzyme-catalyzed
enolizations have values of similar magnitude.[12] Tautomeriza-
tion of a ketone triplet excited state via tunneling has a very
large isotope effect of 700,[13] but the excited singlet is the spe-
cies that is relevant to bioluminescence.
caldehyde catalyst forms a Schiff base with the cyclic amine, la-
bilizing the a-hydrogen and establishing a racemizing equilibri-
um. Tartaric acid forms an insoluble salt with one enantiomer,
removing it from the equilibrium. The salt is hydrolyzed to
give d-(À)-d2-cysteine, which has [a]D À6.58 (c 4.00, 5 n HCl)
([a]D +6.58 (c 4.00, 5 n HCl) for l-cysteine). The conventional
luciferin synthesis was then applied to provide d2-1.
Isotope effects on enzymatic reactions are well known and
heavily studied.[18] They are more complex to consider than
those involved in elementary chemical processes. How they
are measured determines whether they are catalytic rate con-
stant (kcat) isotope effects or kcat/KM isotope effects and what
mechanistic information they provide. However, for both steps
in the bioluminescence mechanism that could be affected by
5,5-d2-luciferin, tautomerization of 4 and formation of 5, the
isotope effect influences the partitioning of an intermediate
between competing pathways, so kH/kD (kinetic isotope effect)
can be considered on this basis.
Another reason to examine the isotope effect with d2-lucifer-
in is the dehydroluciferin side products. Both 5 and 7 are in-
hibitors of firefly luciferase bioluminescence,[14] with inhibition
constant (KI) values of approximately 5 nm and 0.15 mm, re-
spectively; the former being a multi-substrate adduct inhibi-
tor.[15] These compare to a 0.5 mm KI for oxyluciferin[12,16] and a
low mm Michaelis constant (KM) for luciferin. As the dehydrolu-
ciferins are produced to the extent of 20% in each turnover,
their formation contributes substantially to the loss of free,
active enzyme. Their production formally entails b-elimination
of hydrogen peroxide from 3, as proposed by White et al.[2a]
This process could exhibit a substantial isotope effect that
would affect the 4/5 partitioning. Reducing the amount of 3
diverted to 5 and 7 would affect the bioluminescence efficien-
cy and could affect enzyme activity during turnover. Although
we could identify no good precedents for hydrogen peroxide
b-eliminations, conventional b-eliminations often exhibit near-
maximal deuterium isotope effects of approximately 7. Reduc-
ing the formation of dehydroluciferin should increase the unin-
hibited enzyme following each turnover, which should result in
greater integrated bioluminescence and a gentler slope of its
decay. For example, if the isotope effect on dehydroluciferin
formation were 4, d2-1 would cut its production per turnover
from 20% to 6% and double the amount of active enzyme re-
maining after five turnovers.
The first question addressed with d2-luciferin was the iso-
tope effect on bioluminescence. Under identical incubation
conditions with recombinant wild-type P. pyralis luciferase, we
were unable to detect any difference between 1 and d2-1 in
the bioluminescence emission spectrum or intensity.
A second study examined the isotope effect on dehydroluci-
ferin production. The formation of 6 and 7 was determined in
end-point assays. The HPLC analytical method of Silva and co-
workers[4] was used to measure the absolute amounts of oxylu-
ciferin and dehydroluciferin formed from 1 and d2-1, calibrated
with an authentic sample of dehydroluciferin, and their ratio
was used to calculate the isotope effect. Results are summar-
ized in Table 1. From these data, the isotope effect based on
the organic products is 2.13Æ0.14.
Table 1. Production of 6 and 7 from 1 and d2-1.
1
d2-1
6[a]
7[a]
6/7
45.06Æ0.15
11.26Æ0.04
4.002Æ0.01
38.97Æ0.49
4.57Æ0.30
8.53Æ0.56
[a] [nmol].
The d-d2-luciferin required for this study was prepared as
shown in Scheme 2. Commercial d2-cysteine was deracemized
by chiral conversion using the method of Shiraiwa et al.[17] This
process converts Cys to the thiazolidine with acetone. The sali-
A third study was also related to dehydroluciferin produc-
tion, but analyzed the other product. A fluorescence method
based on Amplex Red and horseradish peroxidase[19] was used
to measure hydrogen peroxide formation in triplicate reactions
using 1 or d2-1. The kinetic isotope effect (KIE) determined
from hydrogen peroxide formation is 1.12Æ0.02, which here is
a kcat isotope effect. Reasons for the divergence between this
value and that determined by using dehydroluciferin are not
obvious. However, the data in Table 1 are based on a ratio of
products in the same reaction, which should be more reliable.
The isotope effect on hydrogen peroxide arises from separate
reactions, where consistency between trials is more difficult to
attain.
A final study examined the effect of d2-1 on the time course
of bioluminescence; deuteration was expected to enhance its
Scheme 2. Synthesis of d2-luciferin.
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persistence by reducing the formation of two powerful inhibi-
tors. However, the low magnitude of the isotope effect mutes
the effect compared to the potential benefits earlier discussed.
As shown in Figure 1, there is but a slight advantage for d2-1
Scheme 3. Alternative reaction pathways for the enolate of luciferyl adeny-
late.
Figure 1. Time course of bioluminescence decay at late reaction times for 1
(solid) and d2-1 (dashed).
also be conceived that are consistent with the small isotope
effect on this step.
in bioluminescence decay at long reaction times. Although the
reasons are unclear for the difference in isotope effects on de-
hydroluciferin formation measured via the two reaction prod-
ucts, the conclusion is apparent that the effect is small; this
fact affects mechanistic possibilities for dehydroluciferin
formation.
This view parallels work in bacterial bioluminescent systems,
where a primary kinetic isotope effect of approximately 1.5
was interpreted to show rate-limiting electron transfer to the
carbonyl of its bioluminescent substrate, a long-chain alde-
hyde.[24]
The lack of an isotope effect on oxyluciferin biolumines-
cence emission agrees with earlier beliefs that its keto form is
the emitting species, further dispels outmoded notions that
color modulation of the emission is based on keto-enol tauto-
merism,[20] and shows that CH ionization or tautomerization
are not responsible for its excitation loss.
Acknowledgements
We appreciate financial support from the UCR Vice-Chancellor
for Research (MCP) and Pirrung Consulting. N.D.H. was supported
by a Department of Education Graduate Assistance in Areas of
National Need fellowship (P200A120119). We thank Prof. H. Ai for
access to instrumentation and advice.
The small isotope effect on dehydroluciferin formation is dif-
ficult to reconcile with the idea that it is formed by b-elimina-
tion from 3, as isotope effects on such reactions can be sub-
stantial. We, therefore, consider a modified mechanism for the
reaction of 2 with oxygen, exemplified in Scheme 3 with the
deuterated substrate. As proposed in 1981 by Kosower as well
as more recently[2b,3,21] and then supported experimentally,[22]
the enolate of the adenylate (9) transfers an electron to
oxygen to form radical 10 and superoxide. Their recombination
would produce hydroperoxy d2-3 that cyclizes to the dioxeta-
none. The proportion of bioluminescence and dehydroluciferin
formation could be accounted for by the partitioning of 10 be-
tween recombination with superoxide (which should have only
a very small b-secondary isotope effect) and H/D abstraction
by superoxide. We postulate that the measured isotope effect
reflects the latter reaction, a prototype of which is shown with
a simple ethyl radical 11. We have been unable to locate any
precedent for such a process, and therefore have no frame of
reference for its KIE. Other H-abstractions by superoxide are
known,[23] but their KIEs are not. The idea that dehydroluciferin
arises from elimination of 3 is thus supplanted—instead, it
comes essentially from the direct, step-wise, radical-based oxi-
dation of the enolate of 2 by oxygen. Other mechanisms may
Conflict of Interest
The authors declare no conflict of interest.
Keywords: electron transfer · elimination · isotope effects ·
luminescence · tautomerization
[1] a) V. R. Viviani, Cell. Mol. Life Sci. 2002, 59, 1833–1850; b) J. A. Sundlov,
D. M. Fontaine, T. L. Southworth, B. R. Branchini, A. M. Gulick, Biochem-
istry 2012, 51, 6493–6495.
[2] a) E. H. White, M. G. Steinmetz, J. D. Miano, P. D. Wildes, R. Morland, J.
Am. Chem. Soc. 1980, 102, 3199–3208; b) E. Kosower, Bioluminescence
and Chemiluminescence, Academic Press, San Diego, 1981, p. 365; c) I.
Navizet, D. Roca-Sanjuꢁn, L. Yue, Y.-J. Liu, N. Ferrꢂ, R. Lindh, Photochem.
Photobiol. 2013, 89, 319–325. d) L. Yue, Z. Lan, Y.-J. Liu, J. Phys. Chem.
Lett. 2015, 6, 540–548.
[3] C.-g. Min, A.-m. Ren, X.-n. Li, J.-f. Guo, L. - y. Zou, Y. Sun, J. D. Goddard,
C.-c. Sun, Chem. Phys. Lett. 2011, 506, 269–275.
[4] a) H. Fraga, D. Fernandes, J. Novotny, R. Fontes, J. C. Esteves da Silva,
ChemBioChem 2006, 7, 929–935; b) R. Fontes, B. Ortiz, A. de Diego, A.
Sillero, M. A. Gunther Sillero, FEBS Lett. 1998, 438, 190–194.
ChemistryOpen 2017, 00, 0 – 0
www.chemistryopen.org
3
ꢀ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

[5] a) L. Pinto da Silva, J. C. Esteves da Silva, ChemPhysChem 2011, 12, 951–
960; b) L. Pinto da Silva, R. Simkovitch, D. Huppert, J. C. Esteves da Silva,
ChemPhysChem 2013, 14, 2711–2716; c) L. Pinto da Silva, R. Simkovitch,
D. Huppert, J. C. Esteves da Silva, ChemPhysChem 2013, 14, 3441–3446;
d) M. Rebarz, B.-M. Kukovec, O. V. Maltsev, C. Ruckebusch, L. Hinter-
mann, P. Naumov, M. Sliwa, Chem. Sci. 2013, 4, 3803–3809; e) O. Falklçf,
B. Durbeej, J. Comput. Chem. 2014, 35, 2184–2194; f) A. Ghose, M.
Rebarz, OV. Maltsev, L. Hintermann, C. Ruckebusch, E. Fron, J. Hofkens,
Y. Mꢂly, P. Naumov, M. Sliwa, P. Didier, J. Phys. Chem. B 2015, 119, 2638–
2649; g) Y. Y. Cheng, Y. J. Liu, J. Chem. Theory Comput. 2015, 11, 5360–
5370.
[14] a) C. Ribeiro, J. C. Esteves da Silva, Photochem. Photobiol. Sci. 2008, 7,
1085–1090; b) L. P. da Silva, J. C. Esteves da Silva, Photochem. Photobiol.
Sci. 2011, 10, 1039–1045; c) J. L. Denburg, R. T. Lee, W. D. McElroy, Arch.
Biochem. Biophys. 1969, 134, 381–394; d) B. R. Branchini, R. A. Magyar,
M. H. Murtiashaw, N. C. Portier, Biochemistry 2001, 40, 2410–2418.
[15] N. Thorne, J. Inglese, D. S. Auld, Chem. Biol. 2010, 17, 646–657.
[16] J. M. M. Leit¼o, J. C. G. Esteves da Silva, J. Photochem. Photobiol. B 2010,
101, 1–8.
[17] a) T. Shiraiwa, K. Kataoka, S. Sakata, H. Kurokawa, Bull. Chem. Soc. Jpn.
1989, 62, 109–113; b) T. Shiraiwa, Y. Sado, M. Komure, H. Kurokawa,
Chem. Lett. 1987, 621–622.
[6] Y. Ando, K. Niwa, N. Yamada, T. Irie, T. Enomoto, H. Kubota, Y. Ohmiya,
H. Akiyama, Nat. Photonics 2008, 2, 44–47.
[18] a) D. B. Northrop, Annu. Rev. Biochem. 1981, 50, 103–131; b) W. W. Cle-
land, Arch. Biochem. Biophys. 2005, 433, 2–12.
[7] Y. Erez, I. Presiado, R. Gepshtein, L. Pinto da Silva, J. C. G. Esteves da Sil-
va, D. Huppert, J. Phys. Chem. A 2012, 116, 7452–7461.
[8] O. V. Maltsev, N. K. Nath, P. Naumov, L. Hintermann, Angew. Chem. Int.
Ed. 2014, 53, 847–850; Angew. Chem. 2014, 126, 866–869.
[9] a) F. McCapra, D. J. Gilfoyle, D. W. Young, N. J. Church, P. Spencer, in Bio-
luminescence and Chemiluminescence: Fundamentals and Applied Aspects
(Eds.: A. K., Campbell, L. J., Kricka, P. E. Stanley), Wiley, Chichester, 1994,
p. 387; b) N. J. Church, D. J. Gilfoyle, F. McCapra, P. A. Spencer, D. W.
Young, Synthesis and Applications of Isotopically Labelled Compounds
1994, Proceedings of the International Symposium, 5th, Strasbourg,
1994, 1995, 885–888; c) C. Ganea, V. Vasilescu, in Water and Ions in Bio-
logical Systems (Eds.: A. Pullman, V. Vasilescu, L. Packer), Plenum Press,
New York, 1985, pp. 671–676.
[19] a) V. Mishin, J. P. Gray, D. E. Heck, D. L. Laskin, J. D. Laskin, Free Radical
Biol. Med. 2010, 48, 1485–1491; b) W. G. Gutheil, M. E. Stefanova, R. A.
Nicholas, Anal. Biochem. 2000, 287, 196.
[20] a) E. H. White, R. Rapaport, H. H. Seliger, T. A. Hopkins, Bioorg. Chem.
1971, 1, 92–122; b) E. H. White, D. F. Roswell, Photochem. Photobiol.
1991, 53, 131–136.
[21] D. M. Mofford, G. R. Reddy, S. C. Miller, Proc. Natl. Acad. Sci. USA 2014,
111, 4443–4448.
[22] B. R. Branchini, C. E. Behney, T. L. Southworth, D. M. Fontaine, A. M.
Gulick, DJ. Vinyard, G. W. Brudvig, J. Am. Chem. Soc. 2015, 137, 7592–
7595.
[23] a) E. J. Nanni, Jr., M. D. Stallings, D. T. Sawyer, J. Am. Chem. Soc. 1980,
102, 4481–4485; b) J. M. Fukuto, E. W. Di Stefano, J. N. Burstyn, J. S. Val-
entine, A. K. Cho, Biochemistry 1985, 24, 4161–4167; c) Y. Moro-Oka, P. J.
Chung, H. Arakawa, T. Ikawa, Chem. Lett. 1976, 1293–1296.
[24] W. A. Francisco, H. M. Abu-Soud, A. J. DelMonte, D. A. Singleton, T. O.
Baldwin, F. M. Raushel, Biochemistry 1998, 37, 2596–2606.
[10] B. R. Branchini, M. H. Murtiashaw, R. A. Magyar, N. C. Portier, M. C. Rug-
giero, J. G. Stroh, J. Am. Chem. Soc. 2002, 124, 2112–2113.
[11] a) W. D. Emmons, M. F. Hawthorne, J. Am. Chem. Soc. 1956, 78, 5593–
5596; b) J. Toullec, J. E. Dubois, J. Am. Chem. Soc. 1974, 96, 3524–3532.
[12] X. Liang, P. Talalay, A. S. Mildvan, Biochemistry 1990, 29, 7491–7500.
[13] a) B. Nickel, K. H. Grellmann, J. Stephan, P. Walla, Ber. Bunsen-Ges. 1998,
102, 436–447; b) Y. Pang, W. Chen, in Hydrogen Bonding and Transfer in
the Excited State, Vol. II (Eds.: K.-L. Han, G.-J. Zhao), Wiley, Hoboken,
2011.
Received: August 3, 2017
Version of record online && &&, 2017
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COMMUNICATIONS
Product inhibition: Firefly flashing can
be attributed to inhibitory products of
the luciferase enzyme. A small isotope
effect on the formation of one impor-
tant product, dehydroluciferin, has
mechanistic implications for biolumines-
cence production.
M. C. Pirrung,* A. Dorsey, N. D. Howitt,
J. Liao
&& – &&
b-Deuterium Isotope Effects on Firefly
Luciferase Bioluminescence
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