Photolabile ubiquinone analogues for identification and characterization of quinone binding sites in proteins

Article abstract of DOI:10.1016/j.bmc.2010.03.075

Quinones are essential components in most cell and organelle bioenergetic processes both for direct electron and/or proton transfer reactions but also as means to regulate various bioenergetic processes by sensing cell redox states. To understand how quin

Full text of DOI:10.1016/j.bmc.2010.03.075

Bioorganic & Medicinal Chemistry 18 (2010) 3457–3466
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Photolabile ubiquinone analogues for identification and characterization
of quinone binding sites in proteins
Zhichao Pei a,b, , Tobias Gustavsson b, Robert Roth b,à, Torbjörn Frejd a, Cecilia Hägerhäll b,
*
a Department of Organic Chemistry, Center for Chemistry and Chemical Engineering, Lund University, Box 124, 22100 Lund, Sweden
b Department of Biochemistry and Structural Biology, Center for Chemistry and Chemical Engineering, Lund University, Box 124, 22100 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Quinones are essential components in most cell and organelle bioenergetic processes both for direct
electron and/or proton transfer reactions but also as means to regulate various bioenergetic processes
by sensing cell redox states. To understand how quinones interact with proteins, it is important to have
tools for identifying and characterizing quinone binding sites. In this work three different photo-reactive
azidoquinones were synthesized, two of which are novel compounds, and the methods of synthesis was
improved. The reactivity of the azidoquinones was first tested with model peptides, and the adducts
formed were analyzed by mass spectrometry. The added mass detected was that of the respective azido-
quinone minus N2. Subsequently, the biological activity of the three azidoquinones was assessed, using
three enzyme systems of different complexity, and the ability of the compounds to inactivate the
enzymes upon illumination with long wavelength UV light was investigated. The soluble flavodoxin-like
protein WrbA could only use two of the azidoquinones as substrates, whereas respiratory chain Com-
plexes I and II could utilize all three compounds as electron acceptors. Complex II, purified in detergent,
was very sensitive to illumination also in the absence of azidoquinones, making the ‘therapeutic window’
in that enzyme rather narrow. In membrane bound Complex I, only two of the compounds inactivated the
enzyme, whereas illumination in the presence of the third compound left enzyme activity essentially
unchanged. Since unspecific labeling should be equally effective for all the compounds, this demonstrates
that the observed inactivation is indeed caused by specific labeling.
Received 5 November 2009
Revised 25 March 2010
Accepted 28 March 2010
Available online 3 April 2010
Keywords:
Azidoquinone synthesis
NADH:quinone oxidoreductase
Succinate:quinone oxidoreductase
Flavodoxin
DT-diaphorase
NQO1
Tryptophane repressor binding protein
WrbA
Quinone binding sites
Photolabeling
MALDI-TOF
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the quinone pool. Quinone-like inhibitors are important tools to
better understand the functional mechanism of enzymes using
Quinone binding sites are frequently found in proteins involved
in the bioenergetic processes within cells and organelles. The one
of the most well understood energy coupling mechanism, that of
the bc1 complex or Complex III, occurs entirely via quinone inter-
mediates in the so called Q-cycle,1,2 whereas other membrane-
spanning enzymes such as NADH:quinone oxidoreductase (Com-
plex I3), succinate:quinone oxidoreductase (Complex II4), quinol
oxidase5 and cytochrome bd-oxidase6 contain quinone binding
sites with more or less well characterized properties. Some mem-
brane proteins have a purely regulatory quinone binding site like
the sensor kinase RegB.7 Soluble proteins such as flavodoxin8 and
membrane associated proteins like G3P dehydrogenase9 also
interact with quinone in the membrane, donating electrons to
quinone substrate, but they are also frequently used as insecti-
cides, miticides, piscicides, and herbicides.10–12 Exposure to such
quinone-like inhibitors may cause free radical induced mutations
to mitochondrial DNA that years later lead to the onset of neurode-
generative diseases like Parkinson’s disease.13 Some of the more
serious side effects of anthracycline derived chemotherapeutical
agents like adriamycin and doxorubicin are related to their ability
to act as quinone binding site agonists or antagonists, causing free
radical generation and cardiotoxicity.14–16 At the same time, some
quinone binding proteins are attractive putative drug targets.17 As
more high resolution structural information are becoming avail-
able for bioenergetic enzymes, and our understanding of the func-
tional mechanisms increase concomitantly, there are still many
cases where the exact location and architecture of quinone binding
sites and/or inhibitor binding remains unidentified.
[3H]azidoquinones have been successfully used in the past, to
specifically label quinone binding sites by UV-illumination and
subsequently detect and identify the radiolabeled protein. This
technique, taking advantage of the dark stability and light sensitiv-
ity of photo probes, is becoming an increasingly common tool in
* Corresponding author. Tel.: +46 46 222 0278; fax: +46 46 222 4534.
E-mail address: Cecilia.Hagerhall@biochemistry.lu.se (C. Hägerhäll).
Present address: College of Science, Northwest A&F University, Yangling, Shaanxi
712100, People’s Republic of China
à
Present address: Structural Chemistry Laboratory, AstraZeneca R&D, Peppared-
sleden 1, S-43183 Mölndal, Sweden
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.075

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Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
drug discovery and development in general. A variety of azidoqui-
none compounds have been synthesized over the years, some
showing good activities in some enzymatic systems but not in oth-
ers. For example, in Bos taurus Complex II both membrane anchor
polypeptides were photolabeled with [3H]arylazidoquinone deriv-
atives.18 In subsequent labeling studies using the same enzyme,
two peptide regions, one in SdhC and the other in SdhD, were
assigned as quinone binding sites.19,20 Escherichia coli Complex II
was photo affinity labeled with 3-azido-2-methyl-5-methoxy-
[3H] 6-geranyl-1,4-benzoquinone ([3H]azido-Q).21 The Complex
III quinone binding sites were labeled with 3-azido-2-methyl-
5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone.22 Complex I
from Neurospora crassa mitochondria was labeled with 2,3-
dimethoxy-5-methyl-6-[7-(4-azido-3-[125I] iodophenyl) hepta-
nyl]-p-benzoquinone. This compound functioned as a substrate
for Complex I, and upon illumination one polypeptide was labeled
with high specificity.23 However, the 9.5 kDa labeled polypeptide
corresponded to a subunit not present in the bacterial Complex I,
illustrating the risk of placing the azido group too distantly from
the functionally conserved part of the molecule. Purified E. coli
Complex I was recently labeled with 3-azido-2-methyl-5-methoxy
[3H]-6-decyl-1,4-benzoquinone, that was bound primarily to a
peptide sequence in the NuoM subunit. Labeling of this site was
however only slightly affected by five different Complex I inhibi-
tors,24 demonstrating that in an enzyme complex with more than
one quinone binding site, a particular azidoquinone variant may
specifically label only one of the sites. Direct labeling with various
quinone site inhibitors is also common. Most recently an azido-la-
beled quinazoline-type inhibitor was crosslinked to the 49 kDa
subunit of Complex I from bovine heart25 whereas an acetogenin
inhibitor was used to photo affinity label the ND1 subunit of the
same enzyme.26
Taken together, the direct labeling of quinone binding sites is a
fruitful strategy, but a much greater number of azidoquinone vari-
ants are needed, to be able to target and characterize all putative
binding sites in quinone utilizing proteins. However, the previously
employed methods for synthesis of azidoquinones have been ham-
pered by low or very low yields, limiting the overall availability of
the compounds to the scientific community.
A recent improvement to the original azidoquinone labeling
strategy developed by Chang-An Yu,18 is to detect the labeled pep-
tides with mass spectrometry. Using this approach, the quinone
analogues can be constructed with only one molecular modifica-
tion, the azido group. The feasibility of this approach was recently
demonstrated by Matsumoto et al.6 in labeling the ubiquinol bind-
ing site of cytochrome bd from E. coli.
In this work we describe the construction of three different
photo-reactive quinone analogues, using a safe and more effec-
tive synthesis strategy, and subsequent characterization of the
azidoquinone reactivity using three different enzyme model sys-
tems; a soluble, single polypeptide protein, a membrane protein
complex solubilized from the membrane with detergent, and a
membrane protein complex in situ in the natural membrane.
For effective labeling, the azidoquinone should closely resemble
the natural substrate. The azidoquinone chosen must function
as a substrate for the enzyme to be labeled, and ideally the activ-
ity should also exhibit normal inhibitor sensitivities. The labeling
conditions should also be optimized for each target enzyme, find-
ing the most effective illumination time and quinone:enzyme
stoichiometry.
OH
OH
O
H CO
3
CH
Cl
H CO
3
CH
Cl
H CO
3
CH
3
3
3
a
H CO
3
H CO
3
H CO
3
H
OH
b
O
O
1
O
O
H CO
CH
H CO
CH
Cl
3
3
3
3
c
N
3
H CO
H CO
3
3
O
O
3
2
O
O
O
H C
3
CH
H C
3
CH
H C
3
CH
3
3
3
a, b
c
Cl
N
3
O
O
O
4
5
6
O
O
O
H CO
3
CH
H CO
3
CH
3
H CO
3
CH
3
3
d
e
Br
N
3
H CO
3
H CO
3
H CO
3
O
O
O
1
8
7
Figure 1. Synthesis of AzQ0 (3), DAzB (6), and AzQ1 (8), the two latter being novel compounds. AzQ0 has been synthesized previously, by a less efficient method.22,27 Reagents
and conditions: (a) 37% HCl, H2O, room temperature; (b) 67% HNO3, Et2O, 0 °C, 3 h, 10 °C, 1 h; (c) NaN3, H2O, 95% ethanol, room temperature, 1 h; (d) (NH4)2S2O8, AgNO3,
Br(CH2)5COOH, H2O, CH3CN, 65 °C, 1.5 h; (e) surfactant pillared clay, NaN3, H2O, CHCl3, 95 °C, 3 h.

Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
3459
themselves typically also will be damaged by prolonged UV-irradi-
ation it is important to find conditions were labeling occur but
unspecific damage to the enzyme is minimized. Both azido-com-
pounds reacted in a light-intensity, distance and time dependent
fashion following first order kinetics (Fig. 2). DAzB decomposes
somewhat faster than AzQ0, but this may be explained by that
the parent compound DMB (without azido group) is also more sen-
sitive to UV-illumination than Q0.
The products formed after illumination of AzQ0 and DAzB were
subjected to NMR and HRMS analyses. The former analyses indi-
cated the presence of several different compounds that could not
be assigned with certainty (not shown) whereas in the latter, prod-
ucts with mass corresponding to the azidoquinone minus N2 can
be assigned (See Supplementary data Fig. S1). When higher con-
centrations of azidoquinone were illuminated, gas bubbles were
observed. For AzQ0 the peak of 195.05 correspond to the elemental
composition of C9H9NO4 and the peak of 197.05 correspond to the
elemental composition of C9H11NO4, representing the amino-qui-
none. We tentatively assign the structure of the former product
as corresponding to product 1 and 2 in6 and compounds 3 and 4
in35 (See Supplementary data Fig. S1). For DAzB the same corre-
sponding products were seen (not shown).
2. Results and discussion
2.1. Choice of quinones
The low solubility of the naturally occurring ubiquinone (from
Q6 to Q10) in aqueous solution and the low absorption of quinones
in the visible region has made studies of reaction mechanisms and
protein:quinone interactions more difficult than other similar
reactions. Low molecular weight quinone analogues, especially
those with a reporting group, have offered a promising approach
to study the protein:quinone interaction.22,27 To be able to label
all sites with affinity for the natural ubiquinone, it is desirable to
put the azido group at a position where it is less likely to interfere
with the biological activity. Given the results from the N. crassa
Complex I labeling,23 we also want to avoid having the reacting
azido group located too far away from the quinone head. Therefore,
we have chosen to synthesize ubiquinone derivatives with the azi-
do group either located at a position on the benzoquinone ring, or
at the end of a shorter aliphatic segment (Fig. 1). Since we will use
mass spectrometry to identify the labeled peptides, we can utilize
quinone analogues with only one substituent modification, the azi-
do group.
2.2. Chemistry
1.4
A previously described method for the synthesis of 2,3-dime-
thoxy-5-azido-6-methyl-1,4-benzoquinone (AzQ0)
3 from 2,3-
1.2
dimethoxy-6-methyl-1,4-benzoquinone (Q0) 1, and compound 6
from 4, was based on a direct replacement of a hydrogen atom
on the benzoquinone ring with an azido group under weakly acidic
conditions. The yield was very low (2%)22,27 and the reaction was
difficult to control, often resulting in large amounts of 2,3-dime-
thoxy-5-amino-6-methyl-1,4-benzoquinone being produced. An
alternative strategy was devised by Miyoshi,28 synthesizing 2-azi-
do-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone (azido-ubi-
quinone-2) from a 2-hydroxy-3-methoxy quinone precursor. In
that work the hydroxyl group on the ring was converted to a mesyl
ester and the resulting compound was treated with NaN3 to obtain
the azidoquinone, with an overall yield of 24%.The method we in-
stead used, was to react 1 (or 4) with aqueous HCl to get an addi-
tion product (not isolated), which was subsequently oxidized by
HNO3 to obtain 2 (or 5), followed by substitution of the chloride
for the azide group resulting in 3 (or 6). This reaction can be per-
formed under mild conditions at 0 °C, and the overall yield was
around 50%.29
0 min
5 min
10 min
15 min
1.0
0.8
0.6
0.4
0.2
0.0
240
260
280
300
320
340
360
Wavelength (nm)
0.5
0.0
To synthesize 2,3-dimethoxy-5-(5-azido-pentyl)-6-methyl-
1,4-benzoquinone (AzQ1) (8) using 1 as a starting material, we
first produced 2,3-dimethoxy-5-(5-bromo-pentyl)-6-methyl-1,4-
benzoquinone (Q1-Br) (7) via radical alkylation of the quinone.
The radical was generated from the decarboxylation of the corre-
sponding bromo-acid with silver ion and peroxodisulfate.30,31 This
is a safer method than using SOCl2 and H2O2 to produce the radi-
cal,23,32 since the organic peroxides produced in the latter reaction
decompose rapidly and may explode at high temperature. In the
subsequent step, synthesizing 8 from 7 with sodium azide, it is
particularly important to obtain complete conversion since 8 and
7 have similar polarities making separation difficult. We achieved
a 93% yield using surfactant pillared clay as a phase transfer
catalyst.33,34
-0.5
-1.0
-1.5
-2.0
-2.5
-3.0
-2
0
2
4
6
8
10
12
14
16
Time (minutes)
2.3. Photo-decomposition of the azidoquinones
Figure 2. Bleaching of AzQ by UV-illumination. Optical spectra of AzQ0 (compound
3) during illumination (A) Photodecomposition of AzQ0 over illumination time (B).
0.05 mM AzQ0 in 95% ethanol was placed in a cuvette with 0.2 cm light path and
mounted in an ice bath (0 °C). The sample was continuously illuminated with mid-
wavelength UV light, and optical spectra were recorded with 5 min intervals. DAzB
(compound 6) reacted in an almost identical fashion (not shown).
Photo-decomposition of AzQ0 and DAzB can be conveniently
monitored by optical spectroscopy and thus these compounds
were used to optimize the experimental setup. Since the enzymes

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Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
2.4. Photolabeling of model peptides with azidoquinones
in this peptide, presumably at one of the amines in the side chain.
Aryl azides have previously been shown to react with amines.35
We can not determine if the Asp or the N-terminus are also labeled,
since the a1, b1, and c1 ions are not present in the spectrum. In
addition, immonium ions formed by the His and Tyr could be seen
both in the native and the labeled state (Fig. 4) indicating that the
azido group can in fact react with a wide range of side groups. It
should also be emphasized that many peaks are not assigned,
reflecting the promiscuous reactivity of the azidoquinone. If in-
stead a quinone binding site is labeled, the MSMS analyses will
have a dramatically improved signal to noise ratio.6
To investigate the azidoquinone reactivity with amino acids res-
idues, high concentrations of azidoquinone was illuminated in the
presence of model peptides, and analyzed the resulting products
with mass spectrometry. For AzQ0 labeled peptides we detected
mass shifts of 195.05 0.01 m/z. For AzQ1 labeled peptides we
observed 265.11 0.04 m/z mass shift, and in two cases
248.08 0.04 m/z. The twin peaks in the latter case most probably
result from a de-amidation (Fig. 3). Peptides with two or in some
case up to three AzQ adducts could be detected (Fig. 3A:2 and
not shown). DAzB yielded mass shifts of 149.06 0.03 (not shown).
Recently, Matsumoto et al.,6 used a similar approach as us, but
with a different azidoquinone, for labeling of the quinone binding
site in cytochrome bd. In that work, a glutamate residue was spe-
cifically labeled. In theory, using high concentration of azidoqui-
none and small model peptides instead of protein with a distinct
quinone binding site should yield completely unspecific and ran-
dom labeling. This should permit an investigation of which amino
acid adducts that can be formed. To determine which amino acids
were preferentially targeted, AzQ0 labeled Angiotensin II (with
peptide sequence DRVYIHPF) peptide was subsequently subjected
to MALDI-TOF MSMS to determine which amino acids that had ad-
ducts. The b-ion series showed that the label was predominantly
positioned on the N-terminal amine, Asp or Arg. The y-ion series
support this since y1–y5 were only detected for unmodified pep-
tide while y7 and y8 were seen both with and without adduct
(Fig. 4). This demonstrates that arginine was preferentially labeled
2.5. Choice of biological material for quinone site labeling trials
To test the usefulness of three azidoquinones under different
circumstances, three different model systems of increasing com-
plexity were chosen. As an example of a small, single subunit, sol-
uble protein interacting with quinone we have chosen WrbA, a
tryptophane repressor binding protein in E. coli. WrbA uses FMN
as the sole prosthetic group and belongs to the flavodoxin family,36
and structurally and functionally resembles eukaryotic
NAD(P)H:quinone oxidoreductases.37 WrbA was previously shown
to possess NADH:quinone reductase activity and operates via a
ping–pong mechanism, that is, both NADH and quinone interact
with the same active site.38
Respiratory chain Complex II from Bacillus subtilis is composed
of three protein subunits, a flavoprotein (FP) and an iron–sulfur
protein (IP) protruding from the membrane, and a membrane
Figure 3. (A) Model peptides Angiotensin I (1, with amino acid sequence DRVYIHPFHL), Neurotensin (2, with amino acid sequence pELYENKPRRPYIL), and ACTH (18-39) (3,
with amino acid sequence RPVKVYPNGAEDAEAFPLEF) illuminated in presence of AzQ0 and (B) the same peptides illuminated in presence of AzQ1. The peaks corresponding to
peptide with AzQ adducts are enlarged in A1, A2, A3 and B1, B2, B3. The mass shift seen for the AzQ0 labeled peptides in A is 195.05 0.01 m/z and the mass shift seen for AzQ1
labeled peptides in B is 265.11 0.04 m/z or in two cases 248.08 0.04 m/z. The twin peaks in the latter cases most probably result from a de-amidation.

Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
3461
Figure 4. AzQ0 labeled Angiotensin II (with amino acid sequence DRVYIHPF) subjected to MALDI-TOF MSMS. The star indicate mass of fragments with AzQ0 adduct. The b-ion
series show that the azidoquinone label was predominantly positioned on the aspartate or arginine. The y-ion series also support this since y1–y5 was only detected for
unmodified peptide while y7 and y8 was seen both with and without the adduct. In addition, immonium ions formed by the His and Tyr could be seen both in the native and
the labeled state. The dominant peak in the spectra at 1046.5 corresponds to the peptide that has lost the AzQ0 adduct.
anchor cytochrome b. The FP contains a covalently bound FAD, the
IP harbors three iron–sulfur clusters, and the cytochrome has two
heme groups of b-type.39 In vitro, the enzyme is able to catalyze
both succinate:quinone reductase and quinol:fumarate reductase,
depending on the midpoint potential of the added quinone.40 Most
likely, the enzyme contains two quinone binding sites, the Qp site
located at the interface between IP and the membrane anchor cyto-
chrome, and the Qd site located in the distal end of the membrane
anchor polypeptide.4 The specific inhibitor 2-n-heptyl-4-hydroxy-
quinoline N-oxide (HQNO) blocks 90% or the enzyme, and is known
to bind at the Qd site. The Complex II enzyme was used since it is
membrane spanning, but remains stable and active during purifi-
cation in detergent solubilized state.39
Respiratory chain Complex I from Rhodobacter capsulatus is a
large enzyme complex composed of 14 protein subunits, contain-
ing FMN and eight iron–sulfur clusters as prosthetic groups.41 Se-
ven subunits are membrane spanning and seven subunits form a
large domain protruding from the membrane. The enzyme cataly-
ses the NADH:quinone oxidoreductase reaction coupled to proton
translocation across the membrane by an hitherto unknown mech-
anism.42 Several quinone binding sites are presumably present in
Complex I43 and a number of specific inhibitors, for example rote-
none and piericidin A, interfere with quinone binding.44 However,
the Complex I enzyme is notoriously unstable, and generally looses
its ability to reduce quinones immediately upon addition of deter-
gent.45 Thus, Complex I was chosen to investigate the feasibility
and effectiveness of azidoquinone labeling in situ, in a natural
membrane. This experiment thus offers additional challenges in
term of illumination efficiency in a turbid sample, and the presence
of a protein mixture in the sample.
nient to use in in vitro assays, the corresponding water soluble qui-
none analogues are used. In the test assays we have utilized the
soluble quinones Q0, DMB and Q1 that are commercially available,
and Q0 and DMB were used as starting materials for synthesis of
AzQ0, AzQ1 and DAzB.
2.6.1. WrbA
The activity of purified WrbA protein46 was measured with the
azidoquinones as substrates (Table 1) and the activities were com-
pared to that using the structurally equivalent unmodified quinone
analogues. For AzQ0 and DAzB the activities are virtually the same
as for Q0 and DMB, demonstrating that the azido group did not
interfere with the enzymatic function. The proposed physiological
function of WrbA is to keep the quinone pool reduced under stress
conditions,38 but in fact WrbA has only been demonstrated to react
with soluble quinones. Already when one isoprenoid unit was
present, as in Q1, the activity was almost completely abolished
(
37, Table 1). Thus, it is not surprising that AzQ1 did not work as
a substrate for WrbA.
Subsequently, the WrbA protein was illuminated in the pres-
ence of AzQ0 and DAzB for different time periods, after which the
enzyme activity was measured as before. The azidoquinone con-
tent and illumination time was optimized, with best results ob-
tained at about 5 times molar excess of azidoquinone. The best
illumination time dependent inactivation of the enzyme is shown
in Figure 5. Both azidoquinones inactivated the enzyme, but
AzQ0 was somewhat more effective than DAzB in blocking enzyme
Table 1
WrbA enzyme activity using the different quinone analogues as substrate
2.6. Biological activity and labeling efficiency of the
azidoquinones in the chosen model systems
Vmax (lM/min  mg)
Q0
AzQ0
Q1
AzQ1
DMB
DAzB
684
724
<10
nd
546
464
For effective labeling of quinone binding sites in proteins, it is
important to use an azidoquinone that functions as substrate, pref-
erably resembling the natural substrate as closely as possible. Since
the natural ubiquinones are very hydrophobic and thus inconve-

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Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
100
100
95
90
85
80
75
70
65
DAzB
AzQ0
90
80
70
60
50
SQR:AzQ0 ratio
no addition
1:2
1:5
1:10
1:15
0
1
2
3
4
5
0
1
2
3
4
5
6
7
Illumination time (min)
Illumination time (min)
Figure 5. The NADH:quinone reductase activity of WrbA after photo affinity
labeling with AzQ0 or DAzB. Illuminated samples were collected and kept on ice in
the dark. When the complete sample series had been collected, the activity was
measured immediately. Unspecific inactivation, caused by illumination of sample in
the absence of azidoquinone, has been deducted from the data presented above.
Inactivation in the absence of azidoquinone was less that 5% during 5 min
illumination.
Figure 6. Succinate:quinone reductase activity after photo affinity labeling of
detergent solubilized Complex II purified from B. subtilis using AzQ0. Inactivation in
the absence of azidoquinone, and in the presence of elevated azidoquinone
concentrations are shown to illustrate the narrow window available to monitor
the labeling process in this enzyme. After illumination, the samples where kept on
ice in the dark until the complete sample series had been collected, and then the
activity was measured immediately.
activity. In WrbA, inactivation during illumination in the absence
of azidoquinone was less than 5% during 5 min of constant illumi-
nation, probably since photo-damaged FMN can be exchanged, and
thus replenished, in this enzyme assay (see Section 3.5.1).
none binding sites, but must be bypassing part of the natural elec-
tron transfer pathway. Notably, when we attempted to
compensate for the high unspecific light-dependent inactivation
by adding higher molar ratios of azidoquinone, the inactivation
efficiency instead decreases (Fig. 6). At concentrations above
2.6.2. Complex II
50 lM azidoquinone the quinones start to act as a UV filter, illus-
The enzyme activity of Complex II was measured, both directly
in the membrane-bound state and using the enzyme purified in
detergent, as described in Section 3.4.1. All three azidoquinones
were good substrates for the enzyme, and in the case of the most
water soluble compound AzQ0 and DAzB, the Vmax was even a bit
higher than for the unmodified corresponding quinone (Table 2).
Furthermore, the reactions showed normal inhibitor sensitivity,
with about 10% residual activity seem in the presence of the spe-
cific inhibitor HQNO. We then proceeded to determine the best
labeling conditions for Complex II, in the same way as previously
done for WrbA. The purified, detergent solubilized enzyme was
mixed with different molar ratios of azidoquinone, and the sample
was illuminated. Complex II turned out to be very sensitive to illu-
mination also in the absence of azidoquinone (see Fig. 6). After
5 min of illumination, 25% of the Q1 reductase activity is already
lost, making it cumbersome to observe specific, azidoquinone-in-
duced inactivation, even if specific labeling of the protein may still
occur. Both succinate:quinone reductase activity and succinate
reductase activity to the artificial electron acceptor phenazine
methosulfate (PMS) was affected by illumination, but the latter
activity to a much smaller degree (about 5% decrease after 5 min
of illumination). Succinate:PMS reductase activity is however not
HQNO sensitive,39 and thus this activity does not involve the qui-
trating the importance of carefully optimizing the labeling condi-
tions. Incubation of enzyme with the photo affinity substrates in
darkness did not result in any inhibition (not shown).
2.6.3. Complex I
In Complex I in R. capsulatus membranes, all the soluble qui-
none analogues DMB, Q0 and Q1 were found to be reasonably good
substrates (Table 3). The reactions were sensitive to classical Com-
plex I specific inhibitors, although the maximal inhibition levels
are somewhat lower than for the natural ubiquinone. The azido-
quinones were subsequently evaluated for their ability to act as
electron acceptors from respiratory Complex I during NADH oxida-
tion, using. DMB, Q0 and Q1 as reference compounds as before. As
Table 3
NADH:quinone oxidoreductase enzyme activity and inhibitor sensitivity using the
different quinone analogues as substrate
App. Km
(lM)
Vmax (lmol NADH/(min  mg))
Q0
AzQ0
Q1
AzQ1
DMB
DAzB
75
52
23
21
100
86
0,37
0,58
0,67
0,35
0,35
0,59
Table 2
Imax (% inhibition)
Succinate:quinone oxidoreductase enzyme activity and inhibitor sensitivity using the
different quinone analogues as substrate
Piericidin A
Rotenone
Q0
AzQ0
Q1
AzQ1
DMB
DAzB
68
68
73
79
75
71
48
53
68
79
63
53
Vmax
(lM/min  mg)
Imax (% inhibition) HQNO
Q0
AzQ0
Q1
AzQ1
DMB
DAzB
2.19
3.72
2.52
2.57
3.99
4.72
88%
89%
90%
nd
nd
nd
The maximal inhibition of the NADH oxidase reaction (i.e., using the natural ubi-
quinone in the membrane as electron acceptor) was 92% when using rotenone and
96% when using piericidin A. For experimental details, see Section 3.

Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
3463
seen in Table 3, the azido modifications did not hamper the inter-
100
90
80
70
60
50
action with the enzyme. Just as was seen for Complex II, the azido
modified quinones DAzB and AzQ0 are even better substrates than
the unmodified quinone variants, as demonstrated by the increase
in Vmax and decrease of apparent Km. For AzQ1 there is a 52% de-
crease in Vmax but about the same apparent Km compared to Q1.
However, it must be noted that these two compounds do not only
differ with respect to the azido group, since the isoprenoid chain in
Q1 is substituted for a saturated pentyl group in AzQ1 (Fig. 1). In
bovine Complex I it has been demonstrated that the position of
the methyl group in the isoprenoid tail region of the quinone is rec-
ognized by the enzyme.47 The structural difference might also
influence the partitioning to the hydrophobic phospholipid bilayer
and therefore the local concentration of the substrate at the bind-
ing site. Piericidin A and rotenone are both classical Complex I
inhibitors, acting at the level of quinone reduction, but there is
no consensus on if these inhibitors bind at the same location or
if they target different, distinct quinone binding sites.44 Even so,
the sensitivity to both inhibitors of all the azido compounds are
similar to those of the unmodified quinones (Table 3). Taken to-
gether we conclude that all three azidoquinone analogues accept
electrons from the oxidation of NADH by Complex I in a similar
way to the unmodified ubiquinone analogues. Since the Complex
I enzyme is sensitive to several types of inhibitor compounds we
also tested if any of the products formed from the azido quinones
after illumination were Complex I inhibitors. R. capsulatus mem-
branes were incubated with previously illuminated azidoquinone
and the NADH oxidase activity was measured. The presence of
AzQ0
AzQ1
DAzB
0
1
2
3
4
5
6
Illumination time (min)
Figure 7. NADH:Q1 reductase activity after photo affinity labeling of Complex I in R.
capsulatus membrane vesicles with AzQ0, AzQ1, and DAzB. Activity was measured
immediately after illumination of each individual sample. Unspecific inactivation,
caused by illumination of sample in the absence of azidoquinone, has been
deducted from the data presented above. The NADH:ferricyanide activity of
Complex I did not change during the illumination time interval shown above,
neither in the presence nor in the absence of azidoquinone.
AzQ1 caused an illumination time dependent inactivation of Com-
plex I NADH:Q1 reductase activity but not of NADH:ferricyanide
reductase activity (not shown), demonstrating that a specific phot-
olabeling of the enzyme had occurred. We compared the photola-
beling efficiency at azidoquinone concentrations ranging from
50 lM post-illuminated DAzB had no effect on enzyme activity
2
20
l
M up to 100
lM. The labeling efficiency was slightly higher at
whereas the same amount of photo-reacted AzQ0 or AzQ1 cause a
20% increase in activity. When the same amount of Q0 or Q1 was
added, up to 30% maximal stimulation of activity was seen. This
slight difference most likely reflect the amount of redox competent
quinone remaining in the sample, but in any case we can rule out
that any Complex I inhibitor was formed.
lM azidoquinone, (corresponding to the apparent Km for Q1
and AzQ1 (Table 3)), but began to decrease at higher azidoquinone
concentrations. At higher concentrations the excess azidoquinone
again acted as a UV filter, actually reducing the amount of light
reaching the enzyme and producing specific labeling. To reduce
the amount of unspecific labeling of the enzyme, it is also preferred
to keep the concentration of azidoquinone low. The data shown in
Then, we proceeded with labeling of the R. capsulatus Complex I
in membrane vesicles. The experimental setup was optimized as
before, to minimize unspecific radiation damage of Complex I
and maximize specific labeling in the turbid bacterial membrane
vesicles solution. Since the other respiratory chain enzymes also
may become labeled, and we want to evaluate the effectiveness
of Complex I labeling only, the enzyme activity after illumination
was measured as NADH:Q1 reductase activity rather than
NADH:oxidase activity (see also Section 3). Keeping the sample
on ice during illumination and using a light source to sample dis-
tance of 7 cm we obtained the best results. Under these conditions
the loss of NADH:Q1 reductase activity in a sample without added
azidoquinone was less than 10% after 4 min illumination. At this
time most of the azidoquinone in the sample had reacted. As a con-
trol we also monitored the NADH:ferricyanide reductase activity in
the samples. It is not known if ferricyanide accepts electrons di-
rectly from FMN or from one of the iron–sulfur clusters, but since
the NADH:ferricyanide reductase activity of Complex I is insensi-
tive to rotenone or piericidin A, it is comparable to the previously
described succinate:PMS reductase activity of Complex II, in that it
does not involve the quinone binding sites. The NADH:ferricyanide
reductase activity of the Complex I samples remained constant for
up to 6 min illumination, regardless of if azidoquinone was present
or absent. A pronounced decrease of NADH:ferricyanide reductase
activity does not occur until after more than 20 min illumination
(not shown). This indicates that a particularly UV sensitive func-
tional group, perhaps the unidentified component described in,48
seems to be present between the site of ferricyanide reduction
and the site of quinone pool reduction. Incubation of enzyme with
the photo affinity substrates in darkness showed no inhibition of
NADH:Q1 reductase activity. As seen in Figure 7, both AzQ0 and
Figure 7 were obtained using 20 lM of each of the three azidoqui-
nones. In N. crassa Complex I, the azidoquinone labeling efficiency
was increased in the presence of NADH.23 In our experiment, no
difference in labeling efficiency was seen when the enzyme was
preincubated with 5
sequently illuminated. Addition of 5
20 M of the azidoquinone electron acceptor immediately before
illumination also did not affect the labeling behavior (not shown).
Interestingly, DAzB, that functioned well as a substrate for Com-
plex I (Table 3) and showed even higher light sensitivity than AzQ0,
(not shown) did not inactivate the enzyme. The sample illuminated
in the presence of DAzB (Fig. 7) behaved essentially like a sample
without added azidoquinone. DAzB is indeed the compound that
structurally deviate the most from the natural ubiquinone since
the two methoxy groups in ubiquinone are replaced by a single
methyl in DAzB (Fig. 1 and Table 3). The failure of DAzB to act as
a specific labeling agent must reflect specific features in the archi-
tecture of the binding sites, but also further strengthens the notion
that the other two compounds have indeed targeted the natural
quinone binding sites and labeled the enzyme specifically.
lM NADH (in the presence of KCN) and sub-
lM NADH together with
l
2.7. Conclusion
In this work we have synthesized three azidoquinone ana-
logues, two of which are novel compounds, and tested their ability
to target and specifically label quinone binding sites in different
types of enzymes. All three compounds reacted with model pep-
tides upon illumination, causing mass shifts in the peptides corre-
sponding to the mass of the respective azidoquinone minus N2 gas.

3464
Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
Light-dependent inactivation in the presence of azidoquinones
(0.462 mmol) 2 in 95% ethanol (2 mL), and stirred for 1 h at room
temperature. The reaction mixture was poured onto 7.5 mL of
ice-cold water and extracted with ethyl ether, then dried over
anhydrous MgSO4, and filtered. The solvent was removed under re-
duced pressure to leave the crude product. Compound 3 (yield
80.0%) was obtained after recrystallization with cyclohexane. Melt-
ing point 49–51 °C (27). 1H NMR (CDCl3) d 2.02 (s,3H,CH3), 4.00 (s,
6H,OCH3). IR 2955 cmÀ1 (C–H), 1649 cmÀ1 (C@O), 1610 cmÀ1
(C@C), 2130 cmÀ1 (–N3).
could be seen both using purified, water soluble (Fig. 5) or deter-
gent solubilized enzyme (Fig. 6) and in a turbid membrane vesicle
solution, where the azido-compounds had to compete for the bind-
ing sites with the natural ubiquinone (Fig. 7). In WrbA, only the
two most hydrophilic quinones could be used as substrates,
whereas neither Q1 nor the corresponding AzQ1 showed any en-
zyme activity (Table 1). In Complex II, all three azidoquinones were
as good substrates or better as the corresponding quinone without
the azido group (Table 2), but the labeling efficiency was difficult
to estimate accurately, due to the high degree of UV-induced inac-
tivation of the enzyme also in the absence of azidoquinone (Fig. 6).
Future mass spectrometric analyses will have to reveal the de facto
efficiency of labeling in these samples. In membrane bound Com-
plex I, two of the compounds, AzQ0 and AzQ1, fulfilled the criteria
of being accepted as substrates, showed normal inhibitor sensitiv-
ity and reacted as specific photo-affinity labels. The third com-
pound, DAzB, fulfilled the first three criteria but failed to produce
light-dependent inactivation of Complex I (Table 3, Fig. 7). Since
the three compounds are chemically similar but structurally differ-
ent this finding further strengthens the notion that labeling, specif-
ically targeting the quinone binding sites have occurred in the
other two cases.
3.1.3. 2,6-Methyl-5-azido-1,4-benzoquinone (DAzB) (6)
Compound 6 was synthesized by using the same method as for
the synthesis of 3. The final yield was 20%. 1H NMR (CDCl3) d 1.98
(s, 3H, CH3-2), 2.10 (s, 3H, CH3-6), 6.58 (s, 1H, Ar-H); 13C NMR
(CDCl3) d 10.9, 16.0, 128.0, 131.1, 139.6, 147.3, 182.3, 186.2; IR
2137 cmÀ1 (–N3), 1651 cmÀ1 (C@O), 1617 cmÀ1 (C@C); HRMS
(FAB)) calcd for C8H7O2N3 [M+H]: 177.0538; found: 177.0558.
3.1.4. 2,3-Dimethoxy-5-(5-bromo-pentyl)-6-methyl-1,4-
benzoquinone (Q1-Br) (7)
A solution of 513 mg (2.25 mmol) (NH4)2S2O8 in 2.5 mL water
was added drop wise over 1 h to a stirred suspension of 6 mL
CH3CN and 6 mL water with 273 mg (1.5 mmol) 1, 150 mg AgNO3
and 439 mg (2.25 mmol) bromo-hexanoic acid at 60–65 °C. After
stirring for 20 min, the mixture was cooled to room temperature
and extracted with diethyl ether, then washed with 1 M NaHCO3.
The ether phase was dried over anhydrous MgSO4 and filtered,
then the solvent was removed in vacuo at room temperature. Com-
pound 7 (yield 10%) was obtained after flash chromatography
using pentane/diethyl ether (2:1) as mobile phase. 1H NMR (CDCl3)
d 1.35–1.55 (m, 4H, CH2–CH2), 1.85–1.94 (m, 2H, CH2–(CH2–Br)),
2.03 (s, 3H, Ar-CH3), 2.48 (t, 2H, J = 7.6 Hz, Ar-CH2), 3.42 (t, 2H,
J = 6.7 Hz, CH2–Br), 3.99, 4.00 (s, 6H, 2 Â OCH3). IR; 2938 cmÀ1
(C–H), 1647 cmÀ1 (C@O) 1610 cmÀ1 (C@C), 1262 cmÀ1 (–CH2–).
3. Experimental section
3.1. Chemistry
All chemicals used were of reagent grade. Reactions were rou-
tinely monitored by thin layer chromatography (TLC) on silica gel
plate (F254 Merk), and the products were visualized with UV lamp.
Melting points were determined on a Gallenkamp melting point
apparatus and are uncorrected. 1H and 13C spectra were recorded
with a Bruker Avance 400 instrument or a Bruker DMX 500 instru-
ment at 298 K in CDCl3, using the residual signals from CHCl3 (1H:
d = 7.27 ppm; 13C: d = 77.0 ppm) as internal standard. The infrared
spectra (IR) were recorded on a Shimadzu FT-IR-8300 instrument.
Flash chromatographies were performed using Merk Silica Gel
60 Å. Starting materials 2,3-dimethoxy-6-methyl-1,4-benzoqui-
none (Q0) and 2,6-dimethyl-1,4-benzoquinone (DMB) were pur-
chased from Sigma–Aldrich.
3.1.5. 2,3-Dimethoxy-5-(5-azido-pentyl)-6-methyl-1,4-
benzoquinone (AzQ1) (8)
Montmorillonite (5 g) was first stirred with tetrabutylammo-
nium bromide (3.56 g) in 50 mL water for 120 h at 60–70 °C. The
solution was filtered, and the clay was washed repeatedly with dis-
tilled water and dried in an oven at 100–110 °C over night.34 Com-
pound 7 (16.5 mg, 0.05 mmol) in chloroform (0.5 mL), and sodium
azide (3.9 mg, 0.06 mol) in water (0.5 mL) were mixed in a round-
bottomed flask. The surfactant pillared clay (5 mg) was added, then
the reaction mixture was refluxed with constant stirring for 3 h at
90–100 °C until all the starting material was consumed. The reac-
tion was quenched with water and the product extracted with
chloroform (2 Â 1 mL). The combined extracts were washed three
times with water, dried over anhydrous MgSO4 and filtered. The
solvent was removed under reduced pressure, to obtain 8, an or-
ange oil. The yield was 93%. 1H NMR (CDCl3) d 1.40–1.48 (m, 4H,
CH2–CH2), 1.59–1.68 (m, 2H, CH2-(CH2–N3)), 2.03 (s, 3H, Ar-CH3),
2.48 (t, 2H, J = 6.9 Hz, Ar-CH2), 3.29 (t, 2H, J = 6.8 Hz, CH2–N3),
3.99, 4.00 (s, 6H, 2 Â OCH3). IR 2940 cmÀ1 (C–H), 2096 cmÀ1
(–N3), 1648 cmÀ1 (C@O), 1610 cmÀ1 (C@C), 1266 cmÀ1 (–CH2–).
13C NMR (CDCl3) d 11.9, 26.2, 26.9, 28.2, 28.6, 51.3, 61.2, 138.9,
142.5, 144.3, 184.1, 184.6; HRMS (FAB) calcd for C14H19N3O4
[M+H]: 294.1454; found: 294.1451.
3.1.1. 2,3-Dimethoxy-5-chloride-6-methyl-1,4-benzoquinone
(Q0-Cl) (2)
1.5 mL of 37% hydrochloric acid was diluted with 0.5 mL of H2O
mixed with 1 (1.35 mmol, 245 mg). The mixture was stirred for 3 h
at room temperature, then poured onto 5 mL of ice-cold water and
extracted with diethyl ether. The ether phase was washed with a
saturated sodium chloride aqueous solution until it was neutral,
and then the solvent was removed under reduced pressure. The
residue was taken up with 5 mL ether, cooled to 0 °C and treated
with 0.18 mL HNO3 for 3 h, then stirred for 1 h at 10 °C. Then the
mixture was poured onto 5 mL of ice-cold water and extracted
with diethyl ether. The ether phase was washed with a saturated
sodium chloride solution, dried over anhydrous MgSO4, and then
filtered. The solvent was removed under reduced pressure to get
the orange–red solid. The crude product was purified by recrystal-
lization from cyclohexane to give product 2 (yield 62%). Melting
point 65–67 °C.30 1H NMR (CDCl3) d 2.02 (s, 3H, CH3), 4.02 (s, 6H,
OCH3). IR 2955 cmÀ1 (C–H), 1649 cmÀ1 (C@O), 1610 cmÀ1 (C@C).
3.2. Photodecomposition of azidoquinones
DAzQ and AzQ0 were diluted to 500 lM in 15 mL mH2O and
3.1.2. 2,3-Dimethoxy-5-azido-6-methyl-1,4-benzoquinone
(AzQ0) (3)
illuminated with mid-wavelength UV light for up to 10 min at a
7 cm distance from the light source (UVM-57 (UVP Inc.)). The prod-
ucts formed were collected after evaporating the mH2O and dis-
solved in D2O prior to NMR analysis or dissolved in mH2O prior
A solution containing 60 mg (0.925 mmol) sodium azide in
0.75 mL water was added to
a solution containing 80 mg

Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
3465
to HRMS analysis, performed on a GCT EI+ (Micromass). When
monitoring the photodecomposition by optical spectroscopy DAzQ
or AzQ0 was dissolved to 50 lM in ethanol and illuminated with
ium) and MPYE (complex medium51) resulting in expression of
high amounts of Complex I. The cells were harvested by centrifuga-
tion after reaching the late exponential growth phase. The cells
were washed once in 50 mM Bis–Tris propane, pH 6.8, 20 mM
KCl, 10 mM MgCl2 (Buffer A). Cytoplasmic membranes containing
Complex I was prepared as in,52 except that Buffer A was used in
all steps. The protein concentration was measured by the BCA
method (Pierce) using bovine serum albumin as standard.
the same light source directly in the cuvette.
3.3. Photolabeling and MS analysis of model peptides
Four different commercially available model peptides, Angio-
tensin I, Angiotensin II, Neurotensin and ACTH (18-39), were mixed
with a 200 mol excess of the different AzQs. The mixture was illu-
minated for 4 min on a hydrophobic film placed on ice, at the same
distance from the light source as before. The peptides were dried in
darkness at room temperature, were redissolved in 0.1% TFA, de-
salted and spotted on to a MALDI target plate using Stage Tips as
described in.49 MALDI-TOF MS spectra of the peptides were col-
lected using a 4700 Proteomics Analyzer (Applied Biosystems, Fra-
3.5. Enzyme activity measurement
3.5.1. WrbA activity
WrbA activity was measured in 20 mM potassium phosphate
buffer, pH 7.2, at room temperature. The activity was monitored
as NADH oxidation at 340 nm using
assay was performed in the presence of 100
200 M NADH and 200 M of the respective quinones.
e
NADH = 6.22 mMÀ1 cmÀ1. The
l
M FMN, using
mingham, CA) in reflector mode. Saturated
a
-cyano-4-hydroxy-
l
l
cinnamic acid in 70% acetonitrile and 0.1% TFA was used as matrix.
The spectra were calibrated internally using the known masses of
the model peptides. MSMS spectra were collected in an automated
mode with no collision gas present. The spectra were analyzed
using Data Explorer 4.5 (Applied Biosystems) followed by a manual
interpretation.
3.5.2. Complex II activity
The SQR activity was determined spectroscopically by
monitoring the reduction of DCPIP (2,6-dichlorophenolindophenol)
as the decrease in the absorbance at 600 nm using eDCPIP
=
20.7 mMÀ1 cmÀ1 as described in39 using either phenazine metho-
sulfate (PMS) or the respective quinones as primary electron
acceptor. The following final concentration was used in the assays:
20 mg/mL DCPIP, 20 mM succinate, 3–5 mg SQR and 0,5 mg/mL
PMS or 50 mM quinone. The reactions were done at room temper-
ature in 50 mM potassium phosphate buffer pH 7,4 containing
1 mM potassium cyanide and 0.1 mM EDTA. The reaction was
started by adding PMS or quinone. When indicated, the inhibitor
2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) was added to
3.4. Biological material
WrbA from E. coli, prepared as in,46 was a provided by professor
Jannette Carey. The BCA Protein Assay Kit (Pierce) was used to
determine protein concentrations. The Complex II concentration
was determined spectroscopically using
e
558–575 = 45 mMÀ1 of the
dithionite reduced enzyme.50
10 lM final concentration.
3.4.1. Preparation of B. subtilis membranes and purified enzyme
for Complex II labeling
3.5.3. Complex I enzyme activity
B. subtilis 3G18/pBSD1200, that overproduce Complex II 3–
4 times compared to wild type cells, was grown aerobically in
The NADH:quinone reductase activity was measured as NADH
oxidation at 340 nm
50 mM Tris–Cl, pH 8, 10 mM KCl, 5 mM MgCl2, 0.2
din A, 150 M NADH and 100 M electron acceptor. Apparent ki-
(e
NADH = 6.22 mMÀ1 cmÀ1
)
and 25 °C in
NSMP medium containing 5
lg/mL chloramphenicol at 37 °C and
lg/mL gramici-
harvested in the early stationary growth phase. The cytoplasmic
membrane containing Complex II was prepared from the bacteria
as previously described.39 To purify Complex II, the membranes
were solubilized in dodecylmaltoside (DDM) in 20 mM MOPS at
pH 7.4 over night under gentle stirring, using 5 mg of detergent
per mg of membrane protein. Unsolubilized material was removed
by centrifugation for 1 h at 48,000g. The supernatant was applied
on a DEAE-Sephacel column that had been pre-equilibrated with
20 mM MOPS buffer containing 0.1% (w/v) Thesit. Subsequent
purification steps were done as previously described.39 Peak frac-
tions from ion exchange chromatography were pooled and concen-
trated using Centriprep spin columns with a cut off at 30 kDa. The
sample was diluted in 20 mM MOPS, followed by repeated concen-
tration to reduce the salt concentration. The concentrated sample
was loaded on a S-400 gel filtration column pre-equilibrated with
the MOPS buffer containing 100 mM KCl and 0.1% Thesit. A412 (the
cytochrome b Soret band) and at A280 was measured on the eluted
fractions. The purity and SQR content of the fractions was also esti-
mated from SDS–PAGE gels, before pooling and concentrating the
peak fractions using a Centricon-30 micro concentrator. All purifi-
cation steps were performed at 4 °C and the purified enzyme was
stored on ice in the dark. The Complex II concentration was deter-
l
l
netic constants for the quinones were determined by varying the
quinone concentration (0.2–3 Km) while keeping the concentration
of NADH constant (150 lM). Microcal Origin (Microcal Software,
MA, USA) was used to plot the initial rates and fitting the Michae-
lis–Menten equation to the data for calculation of apparent Km and
Vmax. When measuring inhibition with rotenone the bacterial
membrane vesicles (100
lg protein/mL) were preincubated with
6
lM rotenone at 25 °C for 5 min prior to assay. Piericidin A
(700 nM) was added to the membrane vesicles (4 mg protein /
mL) and the sample was then incubated at 25 °C for 4 min before
the quinone reductase assay. Protein concentration during the
activity measurement was 0.05 mg/mL. Gramicidin (0.2 lg/mL)
was always added to uncouple any transmembrane proton gradi-
ent that may arise during measurements. To rule out that the prod-
ucts formed after illumination of the azido quinones, R. capsulatus
membranes were incubated with 50 lM previously illuminated
AzQs and the NADH:O2 reductase activity was subsequently mea-
sured as before. Activity measurements without additions and
with the corresponding unmodified quinone were performed in
parallel.
mined spectroscopically using
e
558–575 = 45 mMÀ1 and e428–412
=
3.6. Photolabeling reactions
269 mMÀ1 of the dithionite reduced enzyme.
The WrbA sample (10
l
M WrbA and when applicable 50
lM of
3.4.2. Preparation of R. capsulatus membranes for Complex I
the respective azidoquinones in a total volume of 500
l
L 20 mM
labeling
potassium phosphate buffer, pH 7.2) was placed on a concave glass
R. capsulatus (ATCC 17015) was grown aerobically in the dark at
32 °C in baffled E-flasks, in a 1:1 mixture of RCVBN (minimal med-
plate mounted in an ice bath, at a distance of 7 cm from the mid-
wavelength UV light source (UVM-57 (UVP Inc.)). A 100 lL sample

3466
Z. Pei et al. / Bioorg. Med. Chem. 18 (2010) 3457–3466
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was extracted after each different time point of illumination. The
samples were kept on ice in the dark until the entire series was col-
lected. The enzyme activity was measured as described in Section
3.5.1, within 1 h after illumination. Each reaction contained 10
of illuminated enzyme.
lL
The Complex II sample (5
and when applicable from 10 to 75
nones, in a total volume of 500 L 50 mM potassium phosphate
l
M purified Complex II in detergent,
lM of the different azidoqui-
l
buffer with 1 mM EDTA, pH 7.4) was placed on a concave glass
plate mounted in an ice bath, at a distance of 7 cm from the mid-
wavelength UV light source. A 100 lL sample was extracted at each
illumination time point in the series. The samples were kept on ice
in the dark until the entire series was collected. The enzyme activ-
ity was measured within 1 h after illumination as described in Sec-
tion 3.5.2, using Q1 as primary electron acceptor.
Labeling of Complex I containing R. capsulatus membranes was
performed in a quartz cuvette with a 5 mm light path. Cytoplasmic
membranes were diluted in Buffer A to 1 mg/mL protein and pre-
incubated with about 20 lM of the photoactive substrate for
5 min in the dark before illumination. The samples were illumi-
nated with mid-wavelength UV light for up to 10 min at a 7 cm dis-
tance from the light source (UVM-57). The samples were kept at
4 °C throughout the procedure. NADH:quinone reductase activity
was measured immediately after illumination at 340 nm
(e
NADH = 6.22 mMÀ1 cmÀ1) and 25 °C in 50 mM Tris–Cl, pH 8.0,
10 mM KCl, 5 mM MgCl2, 0.2
l
g/mL gramicidin, 5 mM KCN, in
the presence of 100
l
M Q1 and 150 M NADH.
l
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We thank Jannette Carey for supplying purified WrbA. This
work was supported by Grants to C.H. from the Crafoord founda-
tion and Carl Tryggers Foundation and to T.F. from the Swedish Re-
search Council. R.R. acknowledges a scholarship from the Sven and
Lilly Lawski foundation. T.G. is funded by The Research School in
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