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

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

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

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

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

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.

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.

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

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

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

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
.

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

-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

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

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

(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

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-

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

)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

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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