Báo cáo khoa học: Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii Implications for the disulfide metabolism of anaerobic hyperthermophiles doc

Discovery and characterization of a Coenzyme A disulfide
reductase from Pyrococcus horikoshii
Implications for the disulfide metabolism of anaerobic
hyperthermophiles
Dennis R. Harris*, Donald E. Ward
1
, Jeremy M. Feasel
2
, Kyle M. Lancaster
2
, Ryan D. Murphy
2
,
T. Conn Mallet
3
and Edward J. Crane III
2
1 Genencor International, Palo Alto, CA, USA
2 Department of Chemistry, Pomona College, Claremont, CA, USA
3 Center for Structural Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA
While surveying the genomes of hyperthermophilic
and thermophilic Archaea for homologues of the
flavoprotein disulfide reductases, many homologues
with a high degree of identity to the branch of this
family represented by glutathione reductase were
found [1]. Most of the homologues appear to belong
to the subfamily that depend on a redox-active single
cysteine, analogous to the NADH oxidase and per-
oxidase of Enterococcus and the coenzyme A disulfide
reductase (CoADR; EC 1.8.1.14) of Staphylococcus
Correspondence

E. J. Crane III, Department of Chemistry,
Pomona College, 645 North College
Avenue, Claremont, CA 91711, USA
Fax: +1 909 607 7726
Tel: +1 909 607 9634
E-mail:
Website: rwebs.
pomona.edu/ejc14747/EJ_web.htm
*Present address
Department of Biochemistry, University of
Wisconsin-Madison, Madison, WI, USA
(Received 20 July 2004, revised 7 December
2004, accepted 4 January 2005)
doi:10.1111/j.1742-4658.2005.04555.x
We have cloned NADH oxidase homologues from Pyrococcus horikoshii
and P. furiosus, and purified the recombinant form of the P. horikoshii
enzyme to homogeneity from Escherichia coli. Both enzymes (previously
referred to as NOX2) have been shown to act as a coenzyme A disulfide
reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H
+
fi 2CoA-SH +
NAD(P)
+
). The P. horikoshii enzyme shows a k
cat app
of 7.2 s
)1
with
NADPH at 75 °C. While the enzyme shows a preference for NADPH, it is
able to use both NADPH and NADH efficiently, with both giving roughly

equal k
cat
s, while the K
m
for NADPH is roughly eightfold lower than that
for NADH. The enzyme is specific for the CoA disulfide, and does not
show significant reductase activity with other disulfides, including dephos-
pho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H pro-
ceeds in two stages, with an apparent initial reduction of a nonflavin redox
center with the first reduction resulting in what appears to be an EH
2
form
of the enzyme. Addition of a second of NADPH results in the formation
of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is
quite different from the mesophilic staphylococcal version of the enzyme.
This is only the second enzyme with this activity discovered, and the first
from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furio-
sus cells were assayed for small molecular mass thiols and found to contain
0.64 lmol CoAÆg dry weight
)1
(corresponding to 210 lm CoA in the cell)
consistent with CoA acting as a pool of disulfide reducing equivalents.
Abbreviations
CoADR, coenzyme A disulfide reductase (EC# 1.8.1.14); pfCoADR, P. furiosus coenzyme A disulfide reductase; phCoADR, P. horikoshii
coenzyme A disulfide reductase; DTNB, 5,5¢ dithiobis(2-nitrobenzoic acid); EH
2
, two-electron reduced enzyme; EH
4
, four-electron reduced
enzyme; HEPPS, N-(2-hydroxyethyl)piperazine-N¢-3-propanesulfonic acid; NOX, NADH oxidase; NPX, NADH peroxidase; TCA, trichloroacetic

acid.
FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS 1189
aureus [2,3]. These enzymes are proposed to be
involved in the robust oxygen-defense systems of
aerobic and facultatively anaerobic organisms [4,5]
and would not be expected to be present in the
mostly strictly anaerobic hyperthermophiles. Evidence
is mounting, however, for the presence of a vigorous
oxidative stress response in Pyrococcus, including the
discovery of both a novel peroxide-producing super-
oxide reductase [6] and an NADH oxidase [1]. Addi-
tionally, an oxidative stress response has been
characterized in the strictly anaerobic bacterium Clos-
tridium perfringens [7].
Microorganisms in the genus Pyrococcus are strictly
anaerobic hyperthermophiles (T
opt
¼ 100 °C) isolated
from marine hydrothermal vents [8–10]. The genomes
of P. horikoshii, P. furiosus and P. abyssii each contain
at least two NADH oxidase homologues. Characteriza-
tion of one of these homologues (NOX1) from
P. furiosus has been described previously [1]. NOX1
shows a novel H
2
O and H
2
O
2
producing NADH oxid-

ase activity in both the absence and presence of exo-
genous FAD. The second NOX homologue examined
(from P. horikoshii, previously referred to as NOX2)
showed a slow NAD(P)H oxidase activity in the pres-
ence of high concentrations of substrate-level FAD,
i.e., in addition to the enzyme bound FAD. The results
described here demonstrate that this enzyme is not
likely to act as an NADH oxidase in vivo, instead act-
ing as a CoADR. This is only the second demonstra-
ted CoA reductase activity, and the first appearance of
this activity in both the Archaea and in a strict anaer-
obe. While the best known small molecular mass thiol
is probably glutathione, a number of novel thiols such
as mycothiol, c-glutamyl cysteine, and trypanithione
have been found in microorganisms [11]. The function
of these thiols appears to be the maintenance of a
reducing intracellular environment. Due to the pres-
ence of a CoADR homologue in all three pyrococcal
genomes, P. furiosus cells were assayed for the pres-
ence of small molecular mass thiols in order to better
understand the role of this enzyme and thiol ⁄ disulfide
systems in pyrococcal metabolism. The results presen-
ted below provide an insight into the use of a small
molecular mass thiol system for the maintenance of
the internal redox environment in an anaerobic hyper-
thermophile.
Results
Characterization of the recombinant CoADR
The recombinant CoADR from P. horikoshii (phCo-
ADR) was purified 15-fold with a yield of 58% and

a specific activity of 3.26 UÆmg
)1
(oxidase activity)
(Table 1) and 8.3 UÆmg
)1
(CoADR activity). The
phCoADR had a subunit m ¼ 50 k as determined by
SDS ⁄ PAGE, and was shown by both HPLC and con-
ventional size-exclusion chromatography to be a tetra-
meric enzyme of m ¼ 198 k. The enzyme obtained
from the overexpression host is approximately 20%
holoenzyme. After reconstitution with FAD the
enzyme contains 0.92 flavin per subunit based on the
ratio of protein concentration to flavin concentration
(as determined at 460 nm). blast and tfasta analysis
of the phCoADR revealed a significant level of identity
to putative NADH oxidases from hyperthermophiles
and bacterial NADH oxidases from mesophilic sources
(Fig. 1). Of particular interest was the identity to the
well characterized NOXs from mesophilic organisms,
with the highest levels of identity found with the
NADH oxidases from E. faecalis (28%), Streptococcus
mutans (26%), and Brachyspira (Serpulina) hyodysente-
riae (24%). The CoADR from S. aureus is 26% identi-
cal to the P. horikoshii CoADR. As shown in Fig. 1,
the P. furiosus NOX1 and the pf and ph CoADRs con-
tain a cysteine which corresponds to the single redox
active cysteine of the E. faecalis NOX and NPX, as
well as considerable identity in areas that have been
shown to be important for NADH and FAD binding

in these enzymes.
Table 1. Purification of the recombinant coenzyme A disulfide reductase from P. horikoshii. For the purposes of this table, purification was
monitored by the FAD-dependent NADH oxidase activity of the enzyme, with a unit equal to the amount of enzyme required to oxidize
1 lmol NADH in 1 min in the presence of 100 lm NADH, 100 lm FAD, in 50 mm potassium phosphate, pH 7.50 at 75 °C. The specific
activity of the purified enzyme in terms of coenzyme A disulfide reductase activity is 8.3 UÆmg
)1
.
Fraction Total units Total protein (mg)
Specific activity
(unitsÆmg
)1
) Purification (fold) Yield (%)
Crude extract 197 888 0.221 – –
Heat-treated extract 134 77.2 1.73 7.83 68.0
Q-sepharose 120 40.1 2.99 13.5 60.9
Size-exclusion 114 35.0 3.26 14.8 57.8
CoADR from P. horikoshii D. R. Harris et al.
1190 FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS
In order to determine the extinction coefficient of
the enzyme-bound FAD, the FAD was released from
the holoenzyme by trichloroacetic acid (TCA) precipi-
tation of the protein. The e
460
of the enzyme-bound
FAD was determined to be 10 200 m
)1
Æcm
)1
.Itis
interesting to note that attempts to remove the FAD

from the enzyme by treatment in 6.0 m guanidine ⁄ HCl
were unsuccessful, even with overnight incubation at
90 °C. This result is consistent with the extreme stabil-
ity of this enzyme, and consistent with the observation
that thermostable proteins, including the NOX1 from
P. furiosus, are frequently stable in organic solvents
and in the presence of denaturants [1]. The visible
spectrum of the enzyme as purified (Fig. 2) has the
same distinct shoulder in the area of 470 nm as the
mesophilic staphylcoccal enzyme [12].
Fig. 1. Multiple sequence alignment of
P. horikoshii and P. furiosus CoADRs to
known NADH oxidases ⁄ NADH peroxidase ⁄
CoADR. The alignment was performed with
CLUSTAL W. The GenBank accession numbers
for the other enzymes are as follows:
Staphylococcus aureus CoADR (AF041467),
E. faecalis NOX (P37061), E. faecalis NPX
(P37062), P. furiosus NOX (PF_1430634).
D. R. Harris et al. CoADR from P. horikoshii
FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS 1191
CoADR activity of the recombinant P. furiosus
CoADR homologue
The CoADR from P. horikoshii has a homolouge in
P. furiosus that is 92% identical (Fig. 1), indicating
that this gene product was also likely to be a CoADR.
The recombinant P. furiosus CoADR (pfCoADR) was
expressed in E. coli. Heat-treated FAD-reconstituted
extracts of the putative pf CoADR showed a strong
CoADR activity corresponding to a specific activity of

11 UÆmg
)1
of heat-treated extract. This result com-
pares favorably with the specific activity obtained dur-
ing the production of the recombinant CoADR from
P. horikoshii. Like the phCoADR, the pfCoADR is
active with both NADH and NADPH. CoADR activ-
ity was also detected in crude extracts of P. furiosus,
however, the level of activity was low and difficult to
distinguish from background reactions.
Steady-state kinetics of the CoADR and oxidase
reactions
The kinetic constants for the CoADR and oxidase
activities of phCoADR are listed in Table 2. The
enzyme does not show significant NADH peroxidase
activity and is not active in the reduction of glutathi-
one, cystine, or 3 ¢ dephospho-CoA disulfide (unlike the
staphylococcal enzyme, which has a 40 lm K
m
for the
dephospho substrate). The enzyme also does not show
significant 5,5¢ dithiobis(2-nitrobenzoic acid) (DTNB)
reductase in a standard aerobic steady-state kinetic
assay; however, in the anaerobic assays of free thiols
discussed below the enzyme does show DTNB turn-
over with NADPH at a very high concentration of
enzyme (30–60 lm). While NADPH is the preferred
substrate for the CoADR activity based on K
m
, the

two nucleotide substrates have almost identical k
cat
s.
This result is quite distinct from the substrate specifi-
city observed with the staphylococcal enzyme, which
shows a marked preference for NADPH [12].
While phCoADR shows a low level of NAD(P)H
oxidase activity (in the absence of CoA disulfide) in
the absence of substrate level FAD (i.e., FAD added
in addition to that present in the enzyme-bound form),
a significant amount of oxidase activity can be
observed in the presence of additional substrate-level
FAD (Table 2). The k
cat app
obtained in the presence
of 100 lm NADH and FAD and 115 lm O
2
is 8.2 s
)1
,
which correlates well with the catalytic constant
observed for the CoADR reaction.
Thermostability and thermoactivity of the CoADR
phCoADR is stable for months at both )80 °C and
)20 °C, and has half-lives of > 100 and 39 h at 85°
and 95 °C, respectively. Figure 3 shows the dependence
of the oxidase and phCoADR activities on tempera-
ture. While both activities show the preference for high
temperature expected of an enzyme from a hyper-
thermophile, at temperatures above 75 °C both activit-

ies appear to plateau slightly, rather than increasing all
the way to the optimal growth temperature for Pyro-
coccus.
Anaerobic reduction with NAD(P)H and redox
state of the proposed cysteine nonflavin redox
center
As shown in Fig. 2, when phCoADR is titrated anaero-
bically at 60 °C with NADPH the titration shows two
main phases, each corresponding to the addition of
300
0.6
0.4
0.2
0
400 500
Wavelength, nm
0
NADPH, eq
A
600.720
0.040
0.080
123
Absorbance
600 700 800
Fig. 2. Anaerobic reductive titration with NADPH at 60 °C. 56 lM
phCoADR in 50 mM potassium phosphate at pH 7.50 was titrated
with NADPH. Spectra are shown at 0 (—), 1.1 (- - -) and 2.0 (ÆÆÆÆ)
NADPH. Inset, Change in absorbance at 600 (s) and 720 (d)nm
during titration.

Table 2. Michaelis constants for the P. horikoshii CoADR, deter-
mined at 75 °Cin50m
M potassium phosphate, pH 7.50.
Activity-substrate K
m app
(lM) k
cat app
(s
)1
)
CoADR-NADH
a
73 8.1
CoADR-NADPH
a
< 9.0 7.2
CoADR-CoANaCl ⁄ CitoA
b
30 7.1
Oxidase-NADH
c
73 8.2
Oxidase-NADPH
c
13 2.0
Oxidase-FAD
d
22 5.9
a
Determined at 200 lm CoA-S-S-CoA,

b
determined at 100 l m
NADPH,
c
determined at 100 lm FAD,
d
determined at 100 lM
NADH.
CoADR from P. horikoshii D. R. Harris et al.
1192 FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS
roughly one equivalent of NAD(P)H. At 720 nm the
majority of the change occurs during the addition of
the first equivalent of the reducing agent as seen in
Fig. 2 (inset). Little change is observed at 361 nm dur-
ing addition of the first equivalent of NADPH, while
an increase is observed during the addition of the sec-
ond and subsequent equivalents of NADPH. With the
exception of the expected increase at 340 nm from the
addition of free NADPH, little additional change is
seen in the spectra when NADPH is added to a total of
six equivalents. No reduction of the FAD is observed
during addition of excess NAD(P)H. Anaerobic titra-
tion of phCoADR with NADH shows very similar
spectral results to those obtained with NADPH (results
not shown). During anaerobic reduction with dithionite
(data not shown) the FAD becomes reduced upon the
addition of a second equivalent of dithionite, consistent
with initial reduction of a nonflavin redox center, fol-
lowed by reduction of the FAD by the strongly redu-
cing dithionite.

While the spectral changes upon addition of
NADPH appear to correlate well with enzyme states
corresponding to the addition of roughly 1 and 2
equivalents of NADPH, it is difficult to determine
directly from the spectrum the fate of the reduced pyr-
idine nucleotide. During the addition of the first equiv-
alent of NADPH, there is a blue shift in the 380 nm
peak and subsequent small increase in absorbance at
340 nm, although the increase is much less than that
expected for the amount of NADPH added (the
increase corresponds to the addition of 15 lm
NADPH, when 50 lm NADPH had been added).
When the titration is performed at room temperature,
no additional absorbance is observed at 340 nm during
the addition of the first equivalent of NADPH con-
firming that the pyridine nucleotide is consumed during
the addition of the first equivalent (data not shown).
Determination of free thiol content
In order to characterize the redox state of the pro-
posed active site cysteine on the enzyme as purified
and after addition of 0.8 equivalent of NADPH, as
well as any small molecular mass thiols released by the
enzyme, we used the thiol specific reagent DTNB.
Because the enzyme contains only one cysteine residue,
there is only one possible reactive thiol on the enzyme,
in addition to any small molecular mass thiol trapped
in the form of a mixed-disulfide with the cysteine (Cys-
S-S-R). The results of these experiments are shown in
Table 3. The enzyme as purified contains 0.00 equiva-
lents of DTNB reactive thiol. Following anaerobic

reduction with NADPH, less than 0.01 equivalent of
small molecular mass thiol was detected, indicating
that little if any of the enzyme is purified in the mixed
disulfide form. If the NADPH reduced enzyme is kept
anaerobic and assayed for thiol content, 0.85 equival-
ent of thiol is detected, indicating that reduction by
NADPH produces a reactive thiol. If the enzyme is
exposed to air following reduction, this thiol becomes
unreactive, suggesting that it is rapidly oxidized to the
sulfenic acid.
Determination of CoA levels and relative stability
in P. furiosus
To determine whether CoA might play a role as a pool
of reducing equivalents in Pyrococcus, as suggested by
the presence of a CoADR homologue in all three
pyrococcal genomes, cells of P. furiosus were assayed
for small molecular thiols. CoA was present at a con-
centration of 0.64 lmol CoAÆg dry weight
)1
, which
corresponds roughly to 210 lm CoA in the cell (assu-
ming a wet ⁄ dry weight ratio of 3 : 1 [13]). Glutathione
was not detected. CoA was present almost entirely
25
50
100
75
50
25
0

75 100
Temperature
% Activity
Fig. 3. NADH oxidase (d) and CoA disulfide reductase (j) activity
of phCoADR at varying temperatures. Activities at 81.3 (oxidase)
and 85.0 °C (disulfide reductase) were set at 100%.
Table 3. Equivalents of free thiol, as detected by DTNB.
Equivalents
of free thiol
phCoADR, as purified 0.00
NADPH reduced phCoADR, anaerobic 0.85
Small molecular mass thiol
released upon NADPH reduction
0.01
NADPH reduced, exposed to air 0.00
D. R. Harris et al. CoADR from P. horikoshii
FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS 1193
in the thiol form, as there was < 5% detected in the
disulfide form, as determined by the concentration of
(thiol + disulfide)–(thiol).
CoA has previously been shown to be four times
more stable than glutathione and 180 times more sta-
ble than cysteine to the Cu
2+
-catalyzed oxidation to
the disulfide form in the presence of oxygen [14].
Because CoA is significantly more complex than either
cysteine or glutathione, we were interested in determin-
ing whether the increased stability to overoxidation
extends to higher temperatures. For this reason, the

oxidation of thiols at a high temperature was assayed.
It was found that CoA oxidizes approximately 4.4
times slower than glutathione at 88 °Cin50mm imi-
dazole buffer, 1.0 lm Cu
2+
pH 7.50, as shown in
Fig. 4 (under these conditions cysteine oxidizes too
quickly to be measured using conventional techniques).
Under conditions in which the copper and any trace
contaminating metals were chelated with EDTA and
phosphate (50 mm potassium phosphate, 0.50 mm
EDTA, pH 7.50), no significant oxidation of CoA was
observed over 100 min at 88 °C.
Discussion
The phCoADR is only the second CoADR character-
ized to date, and is the first from a hyperthermophile
and Archaeon. Moreover, the enzyme displays distinct
differences from the S. aureus CoADR. The enzyme is
quite distinct from the staphylococcal enzyme in its
ability to use both NADPH and NADH as substrates.
With the S. aureus enzyme, substitution of NADH as
the reducing nucleotide gives approximately 17% of
the rate observed with NADPH [12], while the pyro-
coccal enzyme gives almost identical k
cat
values with
either reduced nucleotide substrate. The K
m app
of < 9
and 73 lm for NADPH and NADH, respectively, do

differ significantly, with NADPH appearing to be the
more efficient substrate.
The reduced nucleotide concentrations have been
determined for P. furiosus [15], and while the total
concentration of NAD(P)(H) was found to be about
half the value seen in the mesophilic Salmonella
typhimurium, the pyrococcal NADP(H) ⁄ NAD(H) ratio
was found to be more than twice that found in
S. typhimurium. This result is not surprising, given the
presence of several unique NADP
+
-dependent cata-
bolic enzymes present in Pyrococcus [15]. The k
cat
of
7.2 s
)1
determined for the phCoADR with NADPH as
the reducing substrate is similar to the k
cat
of 27 s
)1
observed for the staphylococcal enzyme, although it is
lower [12].
Stabilization of enzyme intermediates
Different enzymes within the disulfide reductase family
stabilize different intermediates upon anaerobic reduc-
tion with their nucleotide substrates. The H
2
O-produ-

cing NADH oxidases are reduced by their nucleotide
substrates by four electrons to an EH
4
or EH
4
ÆNAD
+
form, as shown in Scheme 1 [16,17]. In this case the
first reducing equivalent reduces the active site cysteine
residue, which serves as a nonflavin redox center, from
the oxidized sulfenic acid to a reduced thiol species.
This 2e
–
reduced form of the enzyme is referred to as
the EH
2
species [17]. Addition of a second equivalent
of reduced nucleotide substrate results in reduction
of the enzyme-bound FAD, forming the EH
4
or
EH
4
ÆNAD(P)
+
species. In the case of a true oxidase, it
makes sense for the enzyme to stabilize the EH
4
form,
since the reduced flavin is reactive with O

2
.
Other enzymes in the family, such as the NADH
peroxidase or glutathione reductase, tend to stabilize
the enzyme-reduced nucleotide complex referred to
as EH
2
ÆNAD(P)H (Scheme 1) [18,19]. On the EH
2
Æ
NADH complex of the NADH peroxidase from
E. faecalis [20] (Npx: NADH + H
+
+H
2
O
2
fi NAD
+
+2H
2
O), the reducing equivalents of NADH are held
in the nicotinamide ring on the re-side of the flavin
isoalloxazine ring with the C4 position of the nicotina-
mide ring located 3.49 A
˚
from N5 of the isoalloxazine
ring. The active site cysteine thiolate is 3.48 A
˚
from

N5 on the si-side of the flavin, which allows for oxida-
tion of this residue by peroxide, followed by reduction
by NADH via the flavin. This configuration ensures
0
0.4
0.6
0.8
1
100 200 300 400 500
Time (min)
Fraction thiol remaining
Fig. 4. Autoxidation of glutathione (d) and CoA (s) at high tem-
perature (88 °C) in the presence of Cu
2+
. The observed rates of oxi-
dation correspond to pseudo first order rate constants of 5.7 · 10
)3
and 1.3 · 10
)3
min
)1
for glutathione and CoA, respectively.
CoADR from P. horikoshii D. R. Harris et al.
1194 FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS
that NADH is available to be used ‘on demand’ when
a substrate appears while at the same time avoiding
the stabilization of a reduced flavin intermediate that
could react undesirably with O
2
.

In Fig. 2, the redox state of the enzyme-bound FAD
is indicated by the peaks at 380 and 460 nm, with for-
mal reduction and ⁄ or charge transfer to the isoalloxa-
zine ring resulting in a loss of absorbance at these
wavelengths. During the addition of the first equivalent
of NADPH there is a decrease in absorbance at
460 nm with a concomitant increase in long wave-
length (> 510 nm) absorbance. The observed spectral
changes are consistent with the reduction of a nonfla-
vin redox center and the formation of an EH
2
species,
with charge transfer possibly developing between the
FAD and the conserved active site cysteine thiol or
thiolate.
During the anaerobic reduction of the S. aureus
enzyme there is loss of roughly 50% of the absorbance
at 452 nm. In that case, the loss of absorbance is
attributed to an asymmetric reduction of the dimeric
enzyme, with the net result of reduction by the first
equivalent being an oxidized flavin ⁄ oxidized cys resi-
due in the form of a mixed disulfide on one subunit
and a reduced flavin ⁄ reduced cys in the form of a thiol
on the other subunit [12]. The decrease of 460 nm
absorbance during titration of the phCoADR corres-
ponds to at most a 24% loss, which is inconsistent
with the scheme proposed for the S. aureus CoADR.
It is interesting to note, however, that the overall
shape of the titration curve at 452 nm for the sta-
phylococcal enzyme and 460 nm for the phCoADR is

nearly identical, with most of the decrease in absorb-
ance occurring during addition of the first reducing
equivalent.
Addition of a second equivalent of NADPH to the
EH
2
form of phCoADR results in an increase in
absorbance in the regions between 507 and 700 nm
and 400 and 500 nm. There is also an increase and
blue shift in the peak which corresponds to the 380
and 360 nm peaks of the E and EH
2
forms, respect-
ively. This result is inconsistent with reduction of the
FAD and consistent with the formation of an EH
2
Æ
NADPH complex, although further characterization is
currently underway to determine definitively the nature
of this enzyme species. It is apparent, however, that lit-
tle if any of the enzyme is stabilized in the FADH
2
form. It was this finding that led us to originally con-
clude that this enzyme was not likely to act as an
NAD(P)H oxidase in vivo.
The difference in the reductive half reaction, inclu-
ding the apparent lack of subunit asymmetry, and the
difference in quaternary structure (the S. aureus
enzyme is a dimer) suggest that the mesophilic and
thermophilic enzymes, which operate at very different

temperatures, may use different mechanisms. These
differences are currently being investigated.
Redox state of the single cysteine
Based on homology to the single cysteine members of
the disulfide reductase family, it seems likely that the
CoADR uses its single cysteine to catalyze its redox
chemistry. As purified, the CoADR cysteine is not
reactive with DTNB. The observation that the cysteine
becomes reactive following reduction with NADPH,
along with the absence of small molecular mass thiols
released upon reduction, leads to the conclusion that
the enzyme is likely to be purified in the sulfenic acid
form (Scheme 1). This result is not surprising, given
that most of the enzyme is obtained in the apo form
from the overexpression host. It is also not particularly
surprising that this enzyme, whose physiological func-
tion appears to be to serve as a CoADR, is easily
oxidized to the sulfenic acid form, when the anaerobic
nature of Pyrococcus is taken into account. While
Pyrococcus may encounter some oxidative stress, it
seems unlikely that it would be regularly subjected to
Scheme 1. Possible redox states for single-
cysteine containing disulfide reductases.
During reduction from ‘E’ to ‘EH
2
’ the
hydride passes from the NAD(P)H through
the FAD to the nonflavin redox center. An
additional possible species, EH
2

-NAD(P)
+
,is
not shown.
D. R. Harris et al. CoADR from P. horikoshii
FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS 1195
the levels of O
2
present in ambient air. Even if it were,
however, this apparent sulfenic acid state is freely
reducible, so that its production would not seem to
put the enzyme at a disadvantage.
Physiological role of CoADR and NAD(P)H
in Pyrococcus
The maintenance of low intracellular levels of cysteine
in organisms has been attributed to the avoidance of
the hydrogen peroxide produced during the rapid O
2
-
dependent oxidation to cystine, necessitating the use of
other small molecular mass thiols such as glutathione
for the maintenance of internal redox levels. The
results presented above are consistent with a role for
CoA in maintaining a reducing environment or serving
as a pool of reducing equivalents at the very high tem-
peratures and high concentrations of metals found in
the natural environment of Pyrococcus. The observed
CoA concentration of 0.64 lmol CoAÆg dry weight
)1
compares favorably with the result of 1.1 lmolÆg

)1
dry
weight obtained for S. aureus [14], and the correspond-
ing intracellular concentration of 210 lm CoA is well
above the K
m app
of 30 lm for the CoA disulfide. The
pfCoADR (nox A-2 in the annotation of the P. furio-
sus genome) was found to be upregulated more than
fivefold by growth on elemental sulfur (S
0
) [21], a
result which suggests a role for this enzyme in the
eventual transfer of electrons to S
0
. Because CoA lev-
els were determined in cells which were grown in the
absence of S
0
, future studies will determine whether
CoA levels increase during growth in the presence of
S
0
. It is also worth noting that the low CoADR activ-
ity noted in crude extracts of P. furiosus was obtained
from cells grown in the absence of elemental sulfur, so
it seems likely that greatly increased activity will be
seen in extracts of cells grown in the presence of S
0
.

The NAD(P)H dependence of enzymes in the disul-
fide reductase family can be divided into two classes:
(a) enzymes whose function appears to be the main-
tenance of a high R-SH ⁄ R-S-S-R ratio, such as
glutathione reductase, trypanathione reductase and thi-
oredoxin reductase, which tend to be NADPH depend-
ent; and (b) enzymes that appear to be involved more
directly in the reduction of oxidizing compounds and
in the regeneration of oxidized nucleotides for glycoly-
sis, which show a distinct preference for NADH, such
as the NADH oxidase, NADH peroxidase, the NOX1
of P. furiosus, alkyl hydroperoxide reductase, and lipo-
amide dehydrogenase.
The pyrococcal CoADR described in this work is
able to efficiently utilize both NADPH and NADH, a
result which is consistent with the unusual utilization
of reduced nucleotide coenzymes by Pyrococcus. The
central metabolism of this organism uses an unusual
NADPH-dependent sulfide dehydrogenase which is
capable of both the NADPH-dependent reduction of
elemental sulfur and the NADP
+
-dependent oxida-
tion of ferredoxin [22]. A unique NADPH-dependent
alcohol dehydrogenase with wide substrate specificity
and a strong preference for the reduction of alde-
hydes to alcohols is also found in this organism [23].
This unusual mix of NADH and NADPH dependent
reactions in catabolic processes may account for the
finding that the pyrococcal CoADR, which would be

expected to show a strong preference for NADPH, is
able to efficiently utilize either reduced nucleotide
substrate.
Comparison of the phCoADR to the pyrococcal
NADH oxidase
There are now two members of the disulfide reductase
family that have been characterized from Pyrococcus,
the NADH oxidase and the CoADR. These two
enzymes display unique biochemical and enzymatic
properties that are not present in their mesophilic
counterparts. The P. horikoshii CoADR and the
P. furiosus NADH oxidase (NOX1) are 37% identical.
Each appears to play distinct roles in the fermentative
metabolism of Pyrococcus and each is regulated differ-
ently [1,21]. The difference in their reactivity is reflec-
ted in the behavior of these two enzymes in reductive
anaerobic titrations. As shown in Fig. 2 and discussed
above, upon addition of excess NAD(P)H the phCo-
ADR forms what appears to be an EH
2
ÆNAD(P)H
complex with little or no FADH
2
character
(Scheme 1). This can be contrasted to the behavior of
the P. furiosus NOX, which forms what appears to be
an EH
4
ÆNAD
+

complex with a fully reduced FAD
(Scheme 1) upon addition of two equivalents of
NADH (data not shown). In-depth studies comparing
the mechanisms of these two pyrococcal enzymes to
each other and to their mesophilic counterparts will be
presented in a future communication.
Experimental procedures
Growth of microorganisms
P. furiosus (DSM 3638) was grown in a sea salts medium
as previously described [24]. Cellobiose (30 mm), maltose
(10 mm), pyruvate (40 mm), or tryptone (5 gÆL
)1
) was inclu-
ded as primary carbon source. E. coli JM109(kDE3) and
XL-1 were grown at 37 °C in TYP medium [yeast extract
(16 gÆL
)1
), tryptone (16 gÆL
)1
), NaCl (5 gÆ L
)1
), and
CoADR from P. horikoshii D. R. Harris et al.
1196 FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS
K
2
HPO
4
(2.5 gÆL
)1

)]. When required, the following anti-
biotics were included in the medium: kanamycin
(50 lgÆmL
)1
), ampicillin (50 lgÆmL
)1
), and tetracycline
(15 lgÆmL
)1
).
Cloning and expression of the gene encoding
the P. horikoshii and P. furiosus CoADRs
The structural gene encoding the CoADR [previously
referred to as NOX2 (PH0572)] was amplified from P. hori-
koshii or P. furiosus genomic DNA with the oligonucleo-
tides TG100 (5¢-GGCCTCATGAAGAAAAAGGTCGTCA
TAATT-3¢), and TG101 (5¢-GGCCAAGCTTCTAGAAC
TTGAGAACCCTAGC-3¢) (for the P. horikoshii CoADR),
and TG104 (5¢-CGCGCCATGGAAAAGAAAAAGGTA
GTCATAA-3¢) and TG105 (5¢-CGCGGTCGACCTAGAA
CTTCAAAACCCTGGC-3¢) for the P. furiosus CoADR.
The N terminus of the CoADRs were based on the pres-
ence and proper spacing of the ribosome binding site, anno-
tation from the genome sequence, and their good
agreement with the NOX homologues from mesophilic
sources that had been characterized in detail. PCR amplifi-
cation was carried out using Pfu polymerase (Stratagene,
La Jolla, CA), and the resulting 1.3 kb PCR product was
cloned in the NcoI and HindIII sites of pET-24d. The
resulting plasmids, pSMC002 (P. horikoshii) and pSMC004

(P. furiosus) were transformed into E. coli BL21(kDE3).
For production of the recombinant enzymes the strain
JM109 (kDE3) was used.
Production and purification of recombinant
phCoADR
The phCoADR was overexpressed in E. coli JM109(kDE3).
A fermentor growth with 5 L TYP media at 37 °C and
kanamycin (50 lgÆmL
)1
) was started with a 2% inoculation
of overnight culture. When the culture reached D
600
¼ 1.0
( 4.0 h) isopropyl thio-b-d-galactoside was added to a
final concentration of 1.0 mm to induce enzyme production.
The culture was incubated for an additional 4.0 h and the
cells were collected by centrifugation at 8000 g at 4 °C for
15 min. The cells were washed with 50 mm Tris buffer
pH 7.5 and collected by centrifugation (8000 g,4°C,
15 min). The resulting cells ( 20 g) were frozen at )80 °C
until purification.
The thawed cells were resuspended in 50 mm Tris buffer
pH 7.5 (70 mL total volume). Eight-milliliter aliquots of
cell solution were disrupted by 3 · 1 min sonication treat-
ments. Cell debris was removed by centrifugation at
15 000 g and 4 °C for 15 min. The pellet was resuspended
and sonication treatments were repeated (20 mL total vol-
ume) and cell debris was removed by centrifugation
(15 000 g,4°C, 15 min). The supernatant was then incuba-
ted at 85 °C for 10 min and placed on ice. Heat denatured

protein was removed by centrifugation at 15 000 g and
4 °C for 20 min.
Chromatography was performed using an AKTA low-
pressure chromatography system (FPLC) from Pharmacia
Biotech (Piscataway, NJ). The supernatant was applied to a
25 mL Q-Sepharose Hi-Load column (Pharmacia Biotech)
equilibrated with 50 mm Tris buffer pH 7.5 at 3.0 mLÆ
min
)1
. The column was washed with 60 mL 0.1 m KCl
50 mm Tris pH 7.5 to remove excess protein. phCoADR
was eluted by a 120-mL linear gradient from 0.10 to 0.45 m
KCl, with phCoADR eluting between 0.27 and 0.32 m KCl.
Fractions containing phCoADR were pooled and reconsti-
tuted with a final concentration of 1 mm FAD by incuba-
tion at 85 °C for 10 min. The reconstituted pool was then
concentrated and rinsed with 50 mm Tris buffer pH 7.50 by
ultrafiltration with 30 k molecular mass limit filtration
tubes at 3700 g,4°C.
The concentrate (total volume 1.7 mL) was applied to a
120-mL Sephacryl S-200 HR (Pharmacia Biotech) size-
exclusion column equilibrated with 50 mm Tris buffer
pH 7.5 at 0.5 mLÆmin
)1
. Fractions containing phCoADR
were pooled (total volume 20 mL). The procedure yields
35 mg of enzyme with little loss between the steps following
heat treatment ( 30% of activity is lost during the heat
treatment, which is assumed to be due to association of the
enzyme with the large pellet of heat denatured protein

obtained at this stage). The enzyme shows a single band
corresponding to a m ¼ 49 k on SDS ⁄ PAGE (Fig. 5) and
Fig. 5. Coomassie blue stained SDS ⁄ PAGE analysis of the purifica-
tion of the recombinant P. furiosus CoADR. Lane 1, crude extract
(15 lg); lane 2, heat-treated extract (5 lg); lane 3, pooled fractions
from the Q-sepharose column (5 lg); lanes 4 and 5, pooled frac-
tions from the size-exclusion column (1 and 2 lg, respectively). The
left lane contains marker proteins with the indicated subunit
molecular mass (top to bottom): myosin, 195 k; b-galactosidase,
112 k; BSA, 59 k; carbonic anhydrase, 30 k.
D. R. Harris et al. CoADR from P. horikoshii
FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS 1197
gives a single peak by both conventional and HPLC size-
exclusion chromatography. A summary of the purification
is shown in Table 1. Figure 2 shows the UV ⁄ visible spec-
trum of phCoADR, which is typical of a flavoprotein, with
a 280 ⁄ 460 ratio of 11.1.
Production and assay of the recombinant
pfCoADR
pfCoADR was overexpressed in E. coli JM109(kDE3). A
2.8-L Fernbach flask with 325 mL TYP media and kana-
mycin (50 lgÆmL
)1
)at37°C was started with a 2.5%
inoculation of overnight culture. Cells were grown, protein
production was induced, and cells were harvested as des-
cribed above. Cells were disrupted by sonicating for
3 · 30 s on ice, followed by reconstitution with FAD
(1.0 mm)at85°C for 10 min. E. coli proteins that precipi-
tated during the heat treatment were removed by centrifu-

ging in a microcentrifuge for 15 min at 14 000 g.
Standard assays
Standard assays were conducted at 75 °C (cuvette tempera-
ture) in 1.5-mL cuvettes. All substrates, including oxidized
CoA and dephosphoCoA, were from Sigma (St. Louis,
MO). Enzyme and substrate(s) were added to preheated
buffer for a total reaction volume of 840–950 lL. Oxidation
of NAD(P)H was monitored at 340 nm. The standard oxid-
ase assay used to monitor the purification of phCoADR
was conducted with 100 lm NADH and FAD as above. A
unit of CoADR activity is defined as the amount of enzyme
required to reduce 1 lmol of CoA disulfideÆmin
)1
in 50 mm
potassium phosphate pH 7.5, 230 lm CoA disulfide and
100 lm NADPH at 75 °C.
Protein and enzyme-bound FAD extinction
coefficient determinations
Protein concentrations were determined using a modified
Bradford assay (Bio-Rad Protein Assay, Hercules, CA).
The extinction coefficient of the enzyme-bound FAD in
50 mm Tris pH 7.50 was determined using the following
equation: e
460
phCoADR ¼ (A
460
phCoADR) ⁄ [(A
450
of
FAD released from phCoADR in 15% (v ⁄ v) TCA) ⁄ (e

450
FAD in 15% (v ⁄ v) TCA {9750 m
)1
Æcm
)1
})].
Quaternary structure determination
The 120-mL Sephacryl S-200 HR (Pharmacia Biotech) size-
exclusion column was used to determine the quaternary
molecular mass of phCoADR. The column was eluted with
50 mm Tris buffer pH 7.5 at 0.5 mLÆ min
)1
. The native
molecular mass of phCoADR was determined by compar-
ison to standards of mass ¼ 670 k (thyroglobulin), 158 k
(gamma globulin), 44 k (ovalbumin), and 17 k (myoglobin).
This result was confirmed by size exclusion HPLC chroma-
tography on a 300 · 7.8 mm Bio-Rad Bio-Sil SEC-400-5
column.
Determination of enzyme free and released thiols
DTNB was used to detect both protein and small molecular
mass thiols. All determinations used a final concentration
of DTNB of 200 lm . Small molecular mass thiols that
might be in the form of mixed disulfide on the enzyme
(Cys-S-S-R) were determined by reducing the enzyme
(56 lm) in an anaerobic titration with NADPH, followed
by cooling the cuvette on ice, opening the cuvette to air,
and immediately centrifuging the enzyme on a 30 k mole-
cular mass cut-off filter to separate the enzyme from small
molecular mass thiols. The flow-through from the filtration

experiment was assayed for thiol content (providing a
measure of small molecular mass thiols), while the retentate
was rinsed three times with 50 mm potassium phosphate,
0.5 mm EDTA pH 7.5 and also assayed for thiol content
(providing a measure of air-stable, accessible protein thiols).
In order to detect protein thiols that were not stable in air
(such as those that may be converted to sulfenic acids), the
enzyme was reduced by 0.8 equivalents of NADPH in an
anaerobic titration at room temperature (because of a slow
turnover of the enzyme with DTNB, more than one equiv-
alent of NADPH would result in a DTNB reductase activ-
ity), followed by a tip to DTNB. The enzyme as purified
was assayed for reactive thiols simply by adding DTNB, as
above.
Thiol stability assays
Thiol assays were performed using DTNB as a thiol detec-
tion reagent in a modification of a previously published pro-
cedure [25]. An aliquot of the thiol to be assayed (generally
100 lL) was removed and added to 900 lL of assay buffer
containing (final concentrations) 5 mm DTNB, 0.25 m
potassium phosphate, 0.5 mm EDTA pH 7.2. After a 5-min
incubation the absorbance was read at 412 nm. Thiol con-
tent was determined by comparison to a standard curve
determined under the same conditions with an extinction
coefficient of 13 180 m
)1
Æcm
)1
, which is in good agreement
with the previous literature values of 13 600 m

)1
Æcm
)1
[25].
Thermostability and thermoactivity assays
Thermostability assays were performed at )80 °C, ) 20 °C,
85 °C and 95 °C over extended periods of time. An aliquot
of enzyme was placed in a 2.0-mL microcentrifuge tube and
sealed with Parafilm. For lower temperatures, the tube was
placed in the respective freezer and standard activity assays
were performed after 1 month. At higher temperatures, a
CoADR from P. horikoshii D. R. Harris et al.
1198 FEBS Journal 272 (2005) 1189–1200 ª 2005 FEBS
preweighed microcentrifuge tube containing enzyme was set
into a covered beaker along with a damp tissue to prevent
evaporation. Both the mass of the tube and activity of the
enzyme were monitored over time using standard NADH
oxidase assay methods. It was necessary to correct for the
mass of the tube due to concentration of the enzyme solu-
tion from the small amount of evaporation that took place
at high temperature. Thermoactivity assays were done at
temperatures ranging from 30 to 85 °C. Background rates
have been subtracted from each assay at the respective tem-
peratures.
Anaerobic titrations
Anaerobic titrations were conducted as described previously
[26]. The temperature of 60 °C was chosen (rather than
higher temperatures) to minimize the background rate of
NAD(P)H decomposition.
Detection of thiols in P. furiosus

The thiol specific reagent monobromobimane was used to
label cellular thiols, followed by separation via HPLC on
aC
8
column and detection by fluorescence (Farrand Opti-
cal fluorimeter, Valhalla, NY) as described previously
[14,27,28]. Each assay was performed on the cells from
40 mL of culture. Briefly, pelleted cells were resuspended
in 2.5% acetonitrile in 50 mm HEPPS pH 8.0, containing
monobromobimane (2.5 mm) and incubated at 37 °C for
30 min in the dark. Control samples were pretreated with
50 mm N-ethylmaleimde under the same conditions. These
samples were spun briefly in a microcentrifuge and resus-
pended in the above solution. Methane sulfonic acid was
added to 25 mm (final), and suspensions were sonicated
three times for 30 s. Cellular debris was removed by cen-
trifuging in a microcentrifuge for 15 min at 14 000 g, and
the supernatant was analyzed by HPLC. Monobromobi-
mane derivatives of thiol standards were prepared as des-
cribed previously [28]. To determine disulfide content, cell
extracts were treated with dithiothreitol (5 mm) to reduce
the disulfides, and assayed as above. Thiols were quanti-
fied by comparison of peak areas to standard curves of
those standards. Coenzyme A was measured as the sum of
the areas of the CoA and dephospho CoA peaks, as
described previously [14].
Acknowledgements
C. Davis, K. Dorschner and C. Hummel provided
valuable technical support, R. Fahey and G. Newton
provided helpful discussion regarding intracellular thiol

detection methods. This research was supported by an
award from Research Corporation. T.C.M. was sup-
ported by NIH Grant GM35394 to Al Claiborne.
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