Site-specific binding of quinones to proteins through thiol addition and addition-elimination reactions

Article abstract of DOI:10.1021/ja050974x

Ubiquinone-0, menaquinone-0, and 2,3,5-trimethyl-1,4-benzoquinone were site-specifically bound to free cysteine of proteins (yeast iso-1 cytochrome c as a model protein) through thioether bond formation. Model thioether quinone conjugates showed unexpecte

Full text of DOI:10.1021/ja050974x

Published on Web 04/09/2005
Site-Specific Binding of Quinones to Proteins through Thiol Addition and
Addition-Elimination Reactions
Wen-Wu Li,*,† Ju¨rgen Heinze,‡ and Wolfgang Haehnel*,†
Institut fu¨r Biologie II/Biochemie, Scha¨nzlestrasse 1, D-79104 Freiburg, Institut fu¨r Physikalische Chemie,
Albertstrasse 23, Freiburg D-79104, Albert-Ludwigs-UniVersita¨t Freiburg, Germany
Received February 15, 2005; E-mail: wenwu.li@biologie.uni-freiburg.de; haehnel@uni-freiburg.de
Enzymes and biological complexes often utilize noncovalently
or covalently bound cofactors for their functions. Quinones are such
molecules. Three classes of p-quinone: ubi-, mena-, and plastoqui-
none are noncovalently bound components in photosynthetic
electron transfer (ET) and respiratory chains.1 Nature has also
crafted a variety of covalently bound quinone cofactors2 including
a recently found cysteine tryptophylquinone (CTQ).3 CTQ consists
of an o-quinone-modified tryptophan side chain cross-linked to a
cysteine via a thioether bond. These natural constructs are in favor
of generating new functions through chemical modification of pro-
teins with quinones.4 Recently, 3,4-dihydroxy-L-phenylalanine was
incorporated into proteins by expanded genetic-code technique to
generate o-quinone bound to proteins.5 However, is it possible to
simply modify side chains of amino acids with p-quinones to realize
a similar goal? The sulfur addition and substitution reaction to
quinones has been thoroughly investigated,6 which together with
their redox cycling is attributed to one of their toxic mechanisms.7,1b
For instance, 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ-0),8
and 2-methyl-1,4-naphthoquinone (menadione, MQ-0)9 were co-
Figure 1. (A) HPLC chromatograms monitored at 215 nm of reactions
valently bound to the â-93 cysteinyl residue of human oxyhemo-
globin via 1,4-Michael-type of thiol addition to quinones (Scheme
1A). These quinones have also been shown to induce the dimer-
ization of proteins such as ArB sensor kinase via an intermolecular
disulfide bond10 and even the polymerization of the phosphorylated
tubulin binding region of tau protein.11 To better understand the
molecular action mechanisms of these quinones and to provide a
general and simple method of binding quinones to proteins, we
studied the interactions of UQ-0, MQ-0, and 2,3,5-trimethyl-1,4-
benzoquinone (TMBQ) with proteins.
between MQ-0 (solid line), MQ-S(CH2)2OH (dotted line) and cyt c at pH7.5
after 3 h. (B) PSD-MALDI-mass spectrum of MQ-peptide 81-103. (C)
Cartoon structure of yeast iso-1 cyt c (1ycc PDB) drawn with PyMOL.
Cys-102 is modified by quinones via thioether bond formation.
Scheme 1. Covalent Attachment of Quinones to Cysteine of
Proteins via Thiol Addition (A) and Thiol Addition-Elimination
Reaction (B)
Cytochrome c (cyt c) as a common protein electron carrier is
often used to prepare ET models by chemical modification.12 Here
yeast iso-1 cyt c13 from Saccharomyces cereVisiae with free Cys-
102 was used as a model protein. For the coupling reactions, UQ-
0, MQ-0, and TMBQ (dissolved in acetonitrile 1.0 mg/mL, all in
20-fold molar excess) were incubated with cyt c at a concentration
of 1.0 mg/mL (79 µM) in degassed 50 mM Tris-HCl buffer, 100
mM NaCl, pH 7.5. The solutions were then stirred for 2-6 h at
RT under argon in the dark. Reverse phase HPLC of reaction
products between UQ-0, MQ-0 (Figure 1A, solid line), and TMBQ
and cyt c at pH 7.5 shows the formation of the respective quinone
cyt c adducts. (Yields, ESI mass spectra, and ESI-MS data of all
products in Supporting Information.) The chromatograms show also
a disulfide dimer of cyt c and a mixture of unreacted cyt c and
oxidized cyt c with sulfinic acid (SO2H). At pH 4 all reactions
occurred with an increase of quinone-cyt c adducts and a decrease
of disulfide dimer as compared to pH 7.5. The difference masses
between cyt c and UQ-cyt c, MQ-cyt c, TMBQ-cyt c were 180,
167, and 155 ( 1.2, respectively, which indicated that only one
molecule of each quinone was attached. The quinone-modified
proteins were isolated by HPLC.
To further investigate the properties of such thioether quinones,
model compounds were synthesized by coupling of UQ-0, MQ-0,
and TMBQ with 2-mercaptoethanol [HS(CH2)2OH] and/or N-acetyl
L-cysteine [(N-Ac)Cys] using a reductive addition and oxidation
reaction (Supporting Information). Unexpectedly, the oxidized
thioether quinone adducts showed reactivity toward cyt c similar
to that of their parent quinones through a thiol addition-elimination
reaction (Scheme 1B). Figure 1A (dotted line) shows the formation
of the same products as that of MQ-0 by reaction of MQ-S(CH2)2-
OH (an anti-cancer substance)14 with cyt c. This demonstrated the
covalent binding of thioether quinone adduct to protein via a thiol-
exchange reaction, which provided experimental evidence for the
† Institut fu¨r Biologie II/Biochemie.
‡ Institut fu¨r Physikalische Chemie.
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10.1021/ja050974x CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
acid group of Cys.17 The CVs of MQ-0, MQ-(N-Ac)Cys, TMBQ,
and TMBQ-(N-Ac)Cys were similar to those of the UQ derivatives
except for more negative values (Supporting Information). The small
redox shift of E1° is probably due to a modification of the electron
density by sulfur addition. This negative shift of the redox potentials
is consistent with previous data in aqueous solution found for MQ-
(N-Ac)Cys and MQ-0 glutathione conjugates.7,18
Taken together, the electrophilicity of quinones enables their
specific modification of the free Cys-102 of yeast iso-1 cyt c as
that of oxyhemoglobin.8,9 The oxidative property causes the
intermolecular disulfide bond formation as also found for ArB
sensor kinase10 and even polymerization11 of proteins. Through
separation of quinone thioether protein adducts from dimers of
protein the quinones bound to proteins were available. The
generated quinones with a thioether bond possess common structural
features of both noncovalently bound ubi-, mena-, and plastoquinone
and covalently bound CTQ.3 The method provides a general, simple,
and fast route to attach different quinones to cysteine-containing
proteins. Thioether quinone conjugates show addition-elimination
reaction toward thiol groups of proteins, which suggests that these
quinones may be transferred between proteins. These results should
also contribute to the understanding of biological activities, toxicity,
and the anti-cancer mechanism of quinones and thioether quinone
adducts. De novo design and chemical synthesis of proteins with
quinones, heme, and flavin are under investigation for ET studies.19
Figure 2. Normalized UV-vis spectra of UQ-cyt c (dotted line) and cyt
c (solid line) in 50 mM Tris-HCl, 100 mM NaCl, pH 8.0. (Inset) Difference
between spectra.
Acknowledgment. Support from the Volkswagen-Stiftung is
gratefully acknowledged. We thank Bettina Knapp for the measure-
ments of the ESI and PSD-MALDI mass spectra.
Figure 3. Cyclic voltammograms of UQ-0, UQ-S(CH2)2OH, and UQ-(N-
Ac)Cys in CH3CN/0.1 M tetrabutylammonium hexafluorophosphate.
Supporting Information Available: Experimental materials and
details for the synthesis of model compounds, UV-vis spectra of
quinone-cyt c, ESI- and MALDI-PSD mass spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
mechanism of its inhibition of tyrosine phosphatase.14 It also
corrects the conclusion that thioether quinone conjugates are inactive
toward other thiols.15 For ET studies free thiols should be removed
to avoid a possible loss of thioether quinone moiety in proteins.
The specific binding site of the three quinones in cyt c was
investigated by bromide cyanogen cleavage. The peptide fragments
cleaved at Met-80 [sequence 81-103: AFGGLKKEKDRNDLITYL-
KKAC(102)E] containing a quinone moiety were isolated by HPLC.
Post-source decay-matrix assisted laser desorption ionization mass
spectrometry (PSD-MALDI-MS) was used to investigate their
fragmentation. The mass spectrum of the MQ-peptide (m/z 2810)
(Figure 1B) shows two significant fragment ions at m/z 1737 and
1458 which correspond to the y13 ion with a menaquinone moiety
and b13 ion without such modification, respectively. Therefore,
MQ-0 should be located within the last 10 amino acids. In addition,
a significant fragment ion at m/z 2607 can be attributed to the
peptide after loss of menaquinone moiety with a thiol group
(MQSH) via â-elimination.16 This confirmed that MQ-0 was
covalently bound to Cys-102 (Figure 1C). A similar fragmentation
pattern for UQ- and TMBQ-peptide 81-103 indicated the same
binding site as that found with MQ-0.
The UV-vis spectrum of UQ-cyt c (Figure 2) showed in
addition to the absorption of cyt c distinct features with maxima at
268 and 330 nm characteristic for the quinone with thioether linkage
to the protein, as highlighted in the inset.
Changes of redox properties of the quinones by sulfur substitution
were investigated by cyclic voltammetry in acetonitrile. The cyclic
voltammograms (CV) of UQ-0 and UQ-S(CH2)2OH (Figure 3, top
and middle) show two characteristic one-electron reduction steps
with redox potentials E1° and E2° and a minor change of E1° by
the thioether bond from -0.67 to -0.60 V. The CV of UQ-(N-
Ac)Cys (Figure 2, bottom) shows, in addition to the two reduction
steps, an extra one at a more positive potential due to the carboxylic
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