Biochimica et Biophysica Acta 1857 (2016) 380–386

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Biochimica et Biophysica Acta

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Electron transfer between the QmoABC membrane complex and

adenosine 5′-phosphosulfate reductase
Américo G. Duarte ⁎, André A. Santos, Inês A.C. Pereira ⁎
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Estação Agronómica Nacional, 2780-157, Oeiras, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: The dissimilatory adenosine 5′-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction
Received 16 September 2015 pathway that catalyzes the reversible two electron reduction of adenosine 5′-phosphosulfate (APS) to sulfite
Received in revised form 30 December 2015 and adenosine monophosphate (AMP). The physiological electron donor for AprAB is proposed to be the
Accepted 4 January 2016
QmoABC membrane complex, coupling the quinone-pool to sulfate reduction. However, direct electron transfer
Available online 6 January 2016
between these two proteins has never been observed. In this work we demonstrate for the first time direct
Keywords:
electron transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC complex and AprAB. Cyclic
Dissimilatory sulfur metabolism voltammetry conducted with the modified Qmo electrode and AprAB in the electrolyte solution presented the
Respiratory membrane complex Qmo electrochemical signature with two additional well-defined one electron redox processes, attributed to
Electron-transfer the AprAB FAD redox behavior. Moreover, experiments performed under catalytic conditions using the QmoABC
Electrochemistry modified electrode, with AprAB and APS in solution, show a catalytic current peak develop in the cathodic wave,
Quinone-pool attributed to substrate reduction, and which is not observed in the absence of QmoABC. Substrate dependence
conducted with different electrode preparations (with and without immobilized Qmo) demonstrated that the
QmoABC complex is essential for efficient electron delivery to AprAB, in order to sustain catalysis. These results
confirm the role of Qmo in electron transfer to AprAB.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction Dissimilatory AprAB has been purified from different microorgan-

Sulfate reducing prokaryotes (SRP) are widespread in nature [1]
particularly in anaerobic sulfate-rich environments, such as marine
sediments, given their respiratory metabolism that converts sulfate to
sulfide. Although, many decades have been passed since sulfate reduc-
tion was first associated with oxidative phosphorylation [2], the
complete electron transfer pathways and mechanisms for energy
conservation are not fully elucidated. The proteins required for dissim-
ilatory sulfate reduction are highly conserved among SRP and some of
them are also present in sulfur-oxidizing organisms [3]. Sulfate reduc-
tion is an intracellular pathway that requires the transport of sulfate
to the cytoplasm and its subsequent activation, a step catalyzed by the
ATP sulfurylase, producing adenosine 5′-phosphosulfate (APS). APS is
then reduced to sulfite by the adenosine 5′-phosphosulfate reductase
(AprAB), and sulfite is converted to sulfide by the dissimilatory sulfite
reductase (DsrAB) with the involvement of DsrC (Fig. 1A).
Abbreviations: AMP, adenoside monophosphate; APS, adenosine 5′-phosphosulfate;
AprAB, adenosine 5′-phosphosulfate reductase; BN-PAGE, blue native polyacrylamide
gel electrophoresis; CV, cyclic voltammogram; DDM, dodecyl-β-D-maltoside; FAD, flavin
adenosine dinucleotide; KPB, potassium phosphate buffer; QmoABC, quinone-
interacting membrane-bound oxidoreductase; SRP, sulfate reducing prokaryotes.
⁎ Corresponding authors.
E-mail addresses: americoduarte@itqb.unl.pt (A.G. Duarte), ipereira@itqb.unl.pt
(I.A.C. Pereira).
isms [4–9]. It is a flavoenzyme, characterized as an αβ heterodimer,
where the α-subunit (͠75 kDa) comprises the catalytic site, a non-
covalently bound flavin adenosine dinucleotide (FAD) cofactor [9]. The
small β-subunit with approximately 18 kDa harbors two [4Fe4S] clus-
ters, which function in electron transfer [10]. Crystal structures showed
that the catalytic FAD cofactor is close to one of the [4Fe4S] centers
(center I), which is buried inside the β-subunit, whereas the other FeS
cluster (center II), is more solvent exposed and responsible for
accepting electrons from the enzyme physiological electron donor
[7,10,11]. The midpoint redox potentials of the Fe–S clusters were deter-
mined by EPR spectroscopy [5] as −60 mV and −540 mV (vs NHE), for
centers I and II, respectively. The discrepancy between the two redox
potentials was attributed to the different environment surrounding
the Fe–S clusters, where the number of dipoles surrounding the centers
is modulating their redox potential [11] (and references therein). It was
proposed that electrons enter the enzyme by center II and are trans-
ferred to center I, towards the catalytic FAD moiety [7,11]. Substrate dif-
fusion to the buried FAD moiety is assured by a long and wide channel
(approximately 1.7 nm long and 1.0 nm wide), towards the catalytic
site that is partially solvent exposed [7,10]. The physiological electron
donor for AprAB is believed to be the quinone-interacting respiratory
complex, QmoABC, whose genes are often found next to aprAB
(Fig. 1B) [12]. A Desulfovibrio vulgaris qmoABC deletion mutant was un-
able to grow in lactate-sulfate, but remained capable of growing in

http://dx.doi.org/10.1016/j.bbabio.2016.01.001
0005-2728/© 2016 Elsevier B.V. All rights reserved.
 A.G. Duarte et al. / Biochimica et Biophysica Acta 1857 (2016) 380–386
Fig. 1. Schematic representation of the sulfate reduction pathway. A) The complete sulfate
reduction pathway. B) The proposed electron transfer between the quinol pool and the
APS reduction step. Blue arrows indicate electron movement.
lactate-sulfite, demonstrating the essential role of the QmoABC complex
for sulfate, but not sulfite, reduction [13]. Several interaction studies,
including co-immunoprecipitation, surface plasmon resonance, cross-
linking Far-Western blot and tag-affinity purification demonstrated a
physical interaction between QmoABC and AprAB [14]. However, direct
electron transfer between these two proteins was never reported.
The QmoABC complex, is composed by two cytoplasmic subunits
(QmoAB) and one membrane subunit (QmoC). Its biochemical charac-
terization revealed the presence of multiple redox cofactors: two
b-type hemes, two FAD and several FeS centers [12,14]. The QmoC sub-
unit belongs to the family of membrane cytochromes b where its hydro-
phobic C-terminal domain includes six transmembrane helices binding
two heme b groups [12,15]. UV–visible titration revealed the midpoint
redox potentials of the b-type hemes as +75 and −20 mV. Moreover,
using menadiol (a menaquinol analog) as electron donor, it was possi-
ble to completely reduce the hemes, supporting quinol interaction of
the QmoABC complex [12].
In this work we demonstrate for the first time, direct electron
transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC
complex and the soluble AprAB. The QmoABC complex was immobilized
on a pyrolytic graphite electrode, establishing direct electron transfer
with AprAB present in the electrolyte solution, in turnover and non-
turnover conditions. These experiments demonstrate direct electron
transfer between the two proteins, confirming the role of the QmoABC
complex as electron donor for AprAB.
2. Materials and methods
2.1. Cell growth and protein purification
D. desulfuricans ATCC 27774 was grown as previously reported [14].
Both QmoABC complex and AprAB purification were carried in anaero-
bic conditions inside a Coy anaerobic chamber (98% Argon, 2% H2 atmo-
sphere) as previously described by Ramos et al. [14]. The purification
steps were monitored by UV–visible spectroscopy and polyacrylamide
gel electrophoresis (SDS-PAGE for AprAB; and BN-PAGE, for the
QmoABC complex). Protein concentration was estimated by colorimet-
ric assays [16] and a molar extinction coefficient of ε408 nm =
91.4 mM− 1·cm− 1 was estimated for the Qmo complex. The AprAB
concentration was determined by the molar extinction coefficient of
381
ε392 nm = 50 mM−1·cm−1 [8]. Pure protein samples were stored in
100 mM potassium phosphate buffer (KPB) 10% glycerol (and 0.1%
w/v DDM, for QmoABC) at − 80 °C prior to use. The purified enzyme
had a Vmax of 5.8 μmol APS·min−1·mg−1, which is similar to previously
reported [5].
2.2. Electrochemical methods
2.2.1. Protein immobilization to the graphite electrode surface
First, the pyrolytic graphite electrode (∅ = 0.3 cm) was immersed in
a diluted nitric acid solution, rinsed in deionized water, hand polished in
1 μm alumina (for at least 30 min), briefly sonicated and finally rinsed
with deionized water. Drops of protein solution (QmoABC 69 μM and
AprAB 480 μM) from 4 to 8 μl were added to the polished pyrolytic
graphite electrode surface in order to form a protein film using the
solvent casting technique. Electrochemical measurements with AprAB
in solution were made by additions of the protein stock solution to the
electrolyte solution.
2.2.2. Direct electrochemical studies
All electrochemical measurements were conducted inside a Coy an-
aerobic chamber (98% Argon, 2% H2, atmosphere), at room temperature
(23 °C) using an electrochemical cell in a three electrode system: pyro-
lytic graphite electrode as the working electrode, Ag/AgCl electrode as
the reference electrode and a platinum wire as the counter electrode.
Electrodes were connected to a CH potentiostat (Electrochemical ana-
lyzer) with data acquired by the manufacturer's software (CHI1201a).
The electrolyte buffer solution used in all experiments was 250 mM
KPB pH 6.8.
Cyclic voltammetry (CV) was evaluated in different scan rates,
ranging from 5 to 5000 mV s−1 and control experiments were conduct-
ed for all assays with the pyrolytic graphite bare electrode. Subtractions
and voltammogram treatment were conducted with the Origin soft-
ware. In all experiments, the first voltammograms were discarded. All
potentials mentioned in this work were corrected to the Standard
Hydrogen Electrode (SHE), with a 197 mV correction factor.
For non-control diffusion redox processes, the number of electrons
involved was estimated by the signal peak width at half current height
(ΔEp1/2) [17] and the heterogeneous rate constant (ks) was estimated
interpolating the anodic to cathodic peak separation (ΔEp) according
Laviron's formulation [18] (Supporting information).
2.2.3. Electrochemical measurements under turnover conditions
The electrochemical response under turnover conditions was evalu-
ated using the modified QmoABC graphite electrode in the presence of
AprAB in solution. In these experiments increasing amounts of substrate
(APS) were added to the electrolyte buffer from anaerobic stock
solutions. Control experiments were conducted with the bare electrode
with and without AprAB in solution, or with the modified Qmo
electrode without AprAB in solution.
3. Results and discussion
3.1. QmoABC electrochemical characterization
A direct electrochemical response of the immobilized QmoABC com-
plex was observed by the presence of a quasi-reversible redox process
(Fig. 2). This broad signal starts to develop between −160 and 10 mV
in the cathodic wave and between − 60 and 150 mV in the anodic
wave. Subtraction of control voltammograms without protein clearly
evidenced the anodic and cathodic peaks (Fig. 2B) centered at − 9 ±
7 mV. This well–defined redox process presents a current peak intensity
ratio close to one that is dependent with the scan rate increase, as
shown in Fig. 2C, characteristic of a surface confined redox process
[17]. This is the first electrochemical study performed with the QmoABC
complex adsorbed to an electrode surface, showing that it can establish
382 A.G. Duarte et al. / Biochimica et Biophysica Acta 1857 (2016) 380–386

Fig. 2. Cyclic voltammograms of immobilized QmoABC complex (100 mV s−1). A) CV of immobilized QmoABC complex (black line) and control experiment without protein (dashed line);
B) subtraction of the voltammograms in A; C) scan rate dependence of the anodic and cathodic current peak intensities of the QmoABC redox process.

direct electron transfer. Since the QmoABC complex has multiple redox
cofactors (hemes, Fe-S clusters and FAD [12]) with unclear stoichiome-
try and no detailed midpoint redox potential characterization, it is im-
possible to conduct a full deconvolution of the QmoABC redox signal.
The broad signal peak width at half current height (ΔEp1/2) would
suggest one electron is involved in the redox process. However, the
redox process observed should account for the sum of several one or
two electron redox processes arising from the QmoABC cofactors, as
described in the literature for other multi-cofactor proteins [19–21].
The scan rate dependence showed an increase on ΔEp and on the
anodic and cathodic peaks area, more evident at high scan rates
(Table S1 — Supporting information). This may be expected for multi
redox center proteins such as Qmo, since the increase in scan rate will
favor particularly the electron transfer of those redox centers that are

Fig. 3. Direct electrochemical experiments of immobilized and soluble AprAB. All figures show on top the experiment (black) and control (dashed) voltammograms, and below the
respective subtraction. A) CV of the immobilized AprAB (100 mV s−1); B) CV of the immobilized free FAD cofactor (100 mV s−1), Γ = 51 ± 6 pmol·cm−2; C) CV of AprAB in solution
(39 μM, 100 mV s−1).
 A.G. Duarte et al. / Biochimica et Biophysica Acta 1857 (2016) 380–386 383

directly in contact with the electrode surface. Nevertheless, it should be

noted that the potentials where the redox signal starts to develop are in
agreement with the midpoint redox potentials of the QmoABC b-type
hemes (+75 and −20 mV) [12]). This suggests that these redox centers
may be involved in direct electron transfer towards the electrode.

3.2. AprAB electrochemical characterization

The AprAB electrochemical response was evaluated with immobili-

zation to the pyrolytic graphite electrode or in solution (Fig. 3). The
immobilized AprAB electrochemical response showed two close redox
processes, named here as process 1 (at −282 ± 5) and 2 (at −197 ±
4 mV) (Fig. 3A). Process 1 was attributed to the electrochemical
response from the native AprAB, while the much less intense process
2 may arise from an alternative AprAB conformation immobilized at
the electrode surface, such as a minor protein fraction with an exposed
flavin cofactor, since this is not covalently bond to the enzyme. Both the
CV shape and the measured redox potentials are in agreement with
those observed for other flavin-containing proteins [20,22]. The current
peak intensity from both anodic and cathodic processes was propor-
tional to the scan rate increase (supporting information, Fig S1) and
its ratio close to one. Anodic and cathodic peak areas are proportional
and ΔEp remains small (≤67 mV) with scan rate dependence, which is
characteristic of strongly adsorbed redox species at the electrode
surface (Table S1, Supporting information). The measured ΔEp1/2 from
process 1 suggests a one electron redox process is present. However,
the FAD accounts for a two electron redox process (Fig. 3B), so the
broad peak must correspond to the overlap of two one-electron redox
processes. The electrochemical parameters are similar to those observed
for the free FAD cofactor immobilized to the pyrolytic graphite electrode
surface (−317 ± 4 and −220 ± 7 mV, Fig. 3B), suggesting that the two
redox processes observed for the immobilized AprAB are from the direct
electrochemical response of the catalytic FAD. Using process 1 ΔEp and
applying Laviron's formulation [18] (supporting information), the
heterogeneous rate constant for electron transfer (ks) was determined
to be in the order of 4 s−1. The surface coverage was determined to be
51 ± 6 pmol/cm2, which according to the AprAB crystal structures
[7,11], corresponds to a multilayer coating.
When the enzyme is added to the electrolyte solution, the recorded
CVs present only one redox process (Fig. 3C). When AprAB (or free FAD)
is immobilized, the flavin cofactor is orientated towards the electrode,
favoring electron transfer, which is less evident when the enzyme is
maintained in solution. A close examination of the baseline subtracted
voltammograms shows a broad redox peak, developing a shoulder
only in the anodic curve, at higher potential values, while the cathodic
wave presents a broader signal. Therefore, according to what was
observed for the immobilized enzyme, this signal was assigned to the
interaction of the AprAB catalytic FAD with the electrode. The midpoint
redox potential determined was −309 ± 5 mV. The determined ΔEp is
now higher than that observed when the enzyme was immobilized to
the electrode surface. The anodic and cathodic current peak intensity
ratio was close to one and proportional to the scan rate square-root,
which is expected for diffusion controlled systems, since the enzyme
was added to the electrolyte solution (supporting information Fig. S1
and Table S1).
3.3. Direct electron transfer from immobilized QmoABC to AprAB
In order to evaluate direct electron transfer between QmoABC and
AprAB, electrochemical experiments were performed with the modified
QmoABC graphite electrode adding AprAB to the electrolyte solution.
Baseline subtracted voltammograms show two distinct well-defined
redox processes at − 300 ± 7 mV and − 202 ± 6 mV (Fig. 4).
These redox processes are similar to those observed for the direct elec-
trochemical characterization of AprAB either immobilized or in solution
(Section 3.2). The signal resulting from QmoABC reduction (Section 3.1)
Fig. 4. Direct electron transfer between immobilized QmoABC complex and AprAB in
solution. Top — CV with the modified QmoABC electrode with 20 μM AprAB in solution
(50 mV s−1, black line), and control experiment (dashed line); bottom — voltammogram
subtraction.
remains evident from − 110 to 50 mV in the difference voltammo-
grams, besides the redox processes 1 and 2 of AprAB. All these signals
are observed at different scan-rates as shown in supporting information
Fig. S2, where the two well-defined AprAB redox processes become
more evident with scan rate increase (between 50 and 200 mV·s−1).
According to the measured ΔEp1/2, both redox processes of AprAB
account for one electron reduction and the current peak intensity ratio
from the anodic and cathodic peaks is close to one,. However, the anodic
and cathodic current peak intensity is now proportional to the scan rate
increase, as characteristic of surface confined systems whereas for iso-
lated AprAB in solution the current peak intensity is proportional to
the scan rate square root (supporting information Fig. S1 and
Table S1). Additionally, increasing protein concentration of AprAB did
not alter significantly the current peak intensity for both anodic and
cathodic peaks (Supporting information S3). This is consistent with a
physical interaction between the two proteins, producing a QmoABC–
AprAB electron transfer complex, as previously reported [14]. Therefore,
considering the electrode-bound QmoABC as the electron donor unit for
AprAB, it is possible to estimate the heterogeneous rate constant
between the modified QmoABC electrode and the soluble AprAB. The
determined values were in the order of 21 and 68 ± s− 1, for AprAB
processes 1 and 2, respectively. These values are higher than the ones
determined for immobilized AprAB alone and indicate a better interac-
tion and faster electron transfer of AprAB with the modified QmoABC
electrode than with the bare electrode. In order to assure that the ob-
served redox signal is from AprAB interacting with the immobilized
QmoABC complex and not from an unspecific flavin in solution
(e.g. FAD released from denatured enzyme), some free FAD was added
to the electrolyte solution with the QmoABC complex adsorbed to the
working electrode. The obtained CVs (Supporting information, Fig. S4)
show two redox processes quite similar to the results observed for the
immobilized free FAD (Fig. 3B), but dissimilar from those observed
with AprAB.
Electrochemical measurements have been reported as useful tools
to study protein–protein electron transfer, under turnover and non-
384 A.G. Duarte et al. / Biochimica et Biophysica Acta 1857 (2016) 380–386

turnover conditions [23–25]. Our data analysis demonstrate for the first
time direct electron transfer between the immobilized QmoABC
complex and AprAB. When using the bare electrode to transfer elec-
trons to AprAB (immobilized or in solution) the two redox processes
of FAD bound to AprAB appear less resolved than in the presence of
immobilized QmoABC. When QmoABC is adsorbed to the electrode,
electrons are transferred to the AprAB catalytic site through the Qmo
and AprAB FeS centers. The detection of two well-defined one electron
redox processes, suggest a stable flavosemiquinone state in the
presence of QmoABC, a feature observed in AprAB enzymes isolated
from sulfate reducing bacteria [5].
3.4. AprAB catalytic activity with modified QmoABC graphite electrode
Electron transfer between the modified QmoABC electrode and
soluble AprAB was also studied under turnover conditions. For this
purpose, the AprAB catalytic behavior was monitored in the presence
of the APS substrate, with the modified QmoABC graphite electrode as
electron donor. In these experiments a catalytic current was observed,
starting to develop at approximately −150 mV on the cathodic wave
(Fig. 5A1). Baseline voltammogram subtraction produced a defined ca-
thodic catalytic peak, which increased in intensity with higher APS con-
centrations (Fig. 5B1). Since the kinetic studies were conducted with a
stationary pyrolitic graphite electrode, the catalytic current measured de-
pends on substrate diffusion. The described catalytic peak was attributed
to the AprAB catalytic activity of APS reduction. Importantly, absence of
QmoABC from the electrode prevented the production of a catalytic cur-
rent (Fig. 5A2 and B2). The measured current amplitude of the cathodic
current peak shows a substrate concentration dependence (Fig. 5C). The
hyperbolic shape observed with the modified QmoABC electrode is typi-
cal of an enzyme kinetic behavior, showing a maximum current (Imax)
of 0.78 μA and an observed affinity constant (kobs) of 61 μM, which is in
the same order of the KM reported for this enzyme [5]. In contrast, the cat-
alytic activity of AprAB with the bare electrode, without immobilized
QmoABC, showed no significant cathodic current variations (Fig. 5C).
Other control experiments conducted with the bare electrode or the
modified QmoABC electrode without AprAB, using different APS concen-
trations, did not exhibit substantial catalytic current variations (see
Supporting information, Fig. S5).
3.5. Concluding remarks
CVs obtained with the modified QmoABC electrode and AprAB in the
electrolyte buffer, clearly show an electrochemical signal from AprAB
catalytic site, meaning that when AprAB interacts with the adsorbed
Qmo, electrons enter the enzyme and the flavin semi-reduced state is
stabilized. This is supported by the two well-defined redox processes
observed, only when Qmo was immobilized to the electrode (Fig. 4),
which are merged in the free enzyme in solution (Fig. 3). Electrochem-
ical measurements under turnover conditions using the QmoABC

Fig. 5. Direct electron transfer between immobilized QmoABC complex and AprAB, under turnover conditions. A1) Modified QmoABC electrode with AprAB in solution with increasing APS
concentrations; A2) bare pyrolytic graphite electrode with AprAB in solution with increasing APS concentrations; B1) baseline subtracted cathodic wave from modified QmoABC electrode
with AprAB in solution; with increasing APS concentrations; B2) baseline subtracted cathodic wave from bare pyrolytic graphite electrode with AprAB in solution with increasing APS
concentrations; the small black arrows point the minimum and maximum points where current peak intensity was measured. C) Catalytic current of APS reduction measured with
AprAB present in the electrolyte solution and: modified QmoABC electrode (black squares), bare graphite electrode (open squares). The black line corresponds to an enzyme kinetic
hyperbolic plot; Imax = 0.78 μA, kobs = 61 μM. In all experiments: [AprAB] = 5 μM; scan rate — 50 mV s−1.
 A.G. Duarte et al. / Biochimica et Biophysica Acta 1857 (2016) 380–386
modified electrode also demonstrate an effective electron transfer
between the membrane complex and the enzyme, leading to APS reduc-
tion (Fig. 5). Electrons are transferred from the graphite electrode to the
Qmo complex, from where they are correctly orientated to AprAB. Since
QmoA is the subunit proposed to interact with AprAB [14], it is likely to
dock with the AprB subunit since this has the solvent exposed [4Fe4S]
electron transfer center. The midpoint redox potentials of the AprAB
FeS centers and their proximity drives electron transfer towards the cat-
alytic site [7] for APS reduction (Fig. 6A). Additionally, since QmoABC
was characterized as a quinol-oxidizing complex [12], it is proposed
that electrons are transferred from the quinol-pool, through Qmo, to
the AprAB catalytic site.
APS reduction was not detected when the QmoABC complex was ab-
sent from the electrode surface, meaning the electron exchange with
the enzyme catalytic site, observed for isolated AprAB (Fig. 3), does
not sustain catalysis (Fig. 6B). Most likely, when immobilized, AprAB in-
teracts with the electrode in an orientation that favors FAD direct elec-
tron transfer but blocks the enzyme substrate channel [7,10],
preventing substrate diffusion towards the catalytic site. Therefore,
the AprAB catalytic mechanism require that electrons reach the catalytic
FAD site from the adjacent [4Fe4S] center I, and for this electrons must
enter the enzyme via center II. In the present experiments, this electron
transfer mechanism was only accomplished when using the modified
Fig. 6. Electron transfer between the QmoABC complex and dissimilatory AprAB.
A) QmoABC complex immobilized to a pyrolytic graphite surface, transferring electrons
to AprAB for APS reduction (blue arrows). B) Electron transfer between the bare
pyrolytic graphite electrode and AprAB for APS reduction.
385
Qmo electrode, were the AprAB catalytic FAD redox signal presents
two consecutive one electron redox processes (Fig. 4). Physiological
electron transfer between QmoABC and AprAB has been proposed to re-
quire the presence of a third redox partner, in an electron confurcation
process, as direct electron transfer between a menaquinol analog and
AprAB through the Qmo complex has never been observed [14]. By
using this electrochemical strategy, the modified Qmo electrode is capa-
ble of transferring electrons to the soluble AprAB, sustaining catalysis,
which was never observed in solution. This might be explained by
the negative overpotential values imposed by the electrode (below
− 100 mV), that probably drive the reaction, whereas in vivo the
menaquinol pool does not have a low enough redox potential to achieve
reduction of APS, requiring the involvement of a third partner This is the
first electrochemical study conducted with enzymes and respiratory
complexes involved in sulfate reduction. In this work we demonstrate
that there is direct electron transfer between the soluble dissimilatory
AprAB and the membrane QmoABC complex. The proposal of electron
transfer between QmoABC and AprAB was suggested more than ten
years ago but was never demonstrated. By means of electrochemical
measurements it was possible to detect protein–protein electron trans-
fer under turnover and non-turnover conditions. The experiments
conducted under non-turnover conditions show clearly electron
transfer between QmoABC membrane complex and AprAB. Further-
more, this electron transfer supports electrochemical catalytic activity
of AprAB. The electrochemical and kinetic data confirm the role of the
QmoABC complex as electron donor for AprAB.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgments
The authors acknowledge to Ricardo O. Louro and Catarina M.
Paquete for the use of the electrochemical apparatus and anaerobic
chamber, and Antonio L. de Lacey for scientific discussions. This work
was supported by the Fundação para a Ciência e Tecnologia (FCT/
MTCES) through grants PTDC/BBB-BQB/0684/2012 and UID/Multi/
04551/2013, and fellowships SFRH/BPD/84607/2012 (AGD), and SFRH/
BD/77940/2011 (AAS).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbabio.2016.01.001.
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