A novel electron transport system for thermostable
CYP175A1 from Thermus thermophilus HB27
Takao Mandai, Shinsuke Fujiwara and Susumu Imaoka
Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University,
Gakuen, Sanda, Japan
Cytochrome P450s are associated with a number of
physiologically essential reactions, including drug
metabolism, carbon source assimilation, and the bio-
synthesis of steroids, vitamins, prostaglandins, and
antibiotics [1]. Cytochrome P450s have great potential
to perform numerous industrially important reactions.
Indeed, cytochrome P450sca-2 from Streptomyces car-
bophilus has already been used for the production of
pravastatin, a cholesterol-lowering drug [2]. However,
low tolerance to various solvents and high temperature
has generally limited the usefulness of cyto-
chrome P450s for industrial applications. Thermophilic
cytochrome P450s possess extreme stability, and
might be used to overcome such limitations. Recently,
two thermophilic cytochrome P450s, CYP119 and
CYP175A1, were identified in Sulfolobus solfataricus
and Thermus thermophilus, respectively [3,4].
CYP119 is well characterized, and its crystal struc-
ture has been determined in the ligand-free state and
in several ligand-bound states [5,6]. As expected,
CYP119 is highly resistant to both high temperatures
(T
m
=91°C) and high pressures (up to 2 kbar) [7].
Keywords
CYP175A1; ferredoxin; ferredoxin–NAD(P)

m
for NADPH = 4.1 ± 0.2 lm).
Furthermore, the FNR reduced cytochrome c in the presence of NADPH
and Fdx. The T
m
value of the FNR was 99 °C at pH 7.4. With an electron
transport system consisting of Fdx and FNR, CYP175A1 efficiently cata-
lyzed the hydroxylation of b-carotene at the 3-position and 3¢-position at
65 °C, and the K
m
and V
max
values for b-carotene hydroxylation were
14.3 ± 1.6 lm and 18.3 ± 0.6 nmol b-cryptoxanthinÆmin
)1
Ænmol
)1
CYP175A1, respectively. This is the first report of a native electron trans-
port system for CYP175A1.
Abbreviations
Fdx, ferredoxin; FNR, ferredoxin–NAD(P)
+
reductase; IPTG, isopropyl-thio-b-D-galactoside; OFOR, 2-oxoacid:ferredoxin oxidoreductase;
ONFR, oxygenase-coupled NADH–ferredoxin reductase; SD, standard deviation; TR, thioredoxin reductase; UPLC, ultra-performance liquid
chromatography.
2416 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS
The structure of CYP119 exhibits the typical cyto-
chrome P450 fold [5]. However, differences between
CYP119 and other cytochrome P450s include a rela-
tively high number of salt bridges, a low number of

electron transport systems for cytochrome P450s that
cannot be described as belonging to either class I or
class II [1,12]. The electron transport system for
CYP119 is a good example of such a system. In this
case, the electron transport system is composed of
ferredoxin (Fdx) and 2-oxoacid:Fdx oxidoreductase
(OFOR), and utilizes pyruvate as an electron source
rather than NAD(P)H [13,14]. On the other hand, the
native electron transport system for CYP175A1 has
not yet been identified, although the catalytic activity
of CYP175A1 has been detected using an artificial
electron transport system for CYP101 from the meso-
philic bacterium Pseudomonas putida [11].
Most Thermus species are known to produce carot-
enoid-like pigments. CYP175A1 catalyzes the hydrox-
ylation of b-carotene at the 3-position and 3¢-position,
producing zeaxanthin via b-cryptoxanthin [10]. The
zeaxanthin produced by CYP175A1 is used as an inter-
mediate for the synthesis of thermozeaxanthins and
thermobiszeaxanthins, which are the main carotenoids
of T. thermophilus [15]. The insertion of thermozeax-
anthins and thermobiszeaxanthins into the cell mem-
brane reduces membrane fluidity and reinforces the
membrane [16], contributing to the survival of T. ther-
mophilus at high temperatures. Thus, identification of
the electron transport system for CYP175A1 is consid-
ered important not only for developing industrial
applications, but also for investigating the physiologi-
cal characteristics associated with this system.
A native electron transport system for CYP175A1

Then, in order to identify electron transport proteins,
the cytosol of T. thermophilus was separated into five
fractions using an anion exchange column (DE52) by
stepwise elution with KCl (50, 100, 200, 300, and
500 mm). b-Carotene hydroxylation activity was not
detected in the presence of any single fraction, but was
detected in the presence of both the 100 mm KCl and
300 mm KCl fractions with purified CYP175A1 and
NADPH. These results suggest that the electron trans-
port system for CYP175A1 was dependent on
NADPH and composed of at least two proteins in the
100 mm KCl and 300 mm KCl fractions. The 300 mm
KCl fraction from the DE52 column was further puri-
fied using a butyl–Sepharose column and a Mono Q
column. b-Carotene hydroxylation activity was
detected in a major peak when it was reacted with
T. Mandai et al. Thermostable electron transport system
FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2417
purified CYP175A1, NADPH, and the 100 mm KCl
fraction from the DE52 column (data not shown). The
peak was subjected to SDS ⁄ PAGE, and a single band
was observed (Fig. 1A). These purification steps are
summarized in Table 1. The purified protein gave a
UV–visible spectrum with a broad absorption peak at
400 nm and a peak at 280 nm (A
400
⁄ A
280
= 0.63)
(Fig. 1B). The absorption spectrum was very similar to

dimer under nondenaturing conditions. Furthermore,
the protein encoded by TTC0096 gave a UV–visible
spectrum with absorption peaks at 273, 392, and
473 nm, which is characteristic of flavoproteins
(Fig. 2B). The FAD content of the protein was
0.70 mol FADÆmol
)1
subunit, suggesting that the FAD
was noncovalently bound to the protein. These results
suggest that another component of an electron trans-
port system for CYP175A1 is a protein encoded by
TTC0096, which functions as an FNR. Thus, we
concluded that the electron transport system for
CYP175A1 belongs to class I.
Characterization of recombinant FNR
The FNR and Fdx were expressed in Escherichia coli
and purified to homogeneity. The purified recombinant
FNR and Fdx had the same chromatographic, photo-
metric and catalytic properties as the native FNR and
Fdx (data not shown). Although the FNR reduced ferri-
cyanide, an artificial electron acceptor, at 25 °C and at
pH 7.4 in the presence of NADH as well as NADPH,
the K
m
value of the FNR for NADPH was about
600-fold lower than that for NADH, and the V
max
value
of the FNR with NADPH was about 55-fold higher
A

than that with NADH (Table 3). Taken together, these
results show that the FNR prefers NADPH over
NADH. Furthermore, the FNR showed 4.2-fold greater
ferricyanide reduction activity at 50 °C with saturating
concentrations of NADPH (1 mm) and ferricyanide
(1 mm) than at 25 °C (data not shown).
To determine the optimal pH of the FNR, we mea-
sured ferricyanide reduction activity at 50 °C and at a
range of pH values from 4.0 to 8.0 (Fig. 3A).
Although the intracellular pH of T. thermophilus is
known to be maintained at 6.9–7.1 [20], the FNR
unexpectedly exhibited maximal activity at pH 4.5–6.5.
The thermostability of the FNR was evaluated by
measuring the residual ferricyanide reduction activity
after incubation of the FNR for 30 min at various
temperatures (Fig. 3B). The T
m
values of the FNR at
pH 7.4 and at pH 5.0 were 99 and 95 °C, respectively.
These results indicate that the FNR is an extremely
thermostable protein at both pH 7.4 and pH 5.0.
The FNR reduced cytochrome c at 50 °C in the
presence of NADPH and Fdx, and the activity was
dependent on the concentration of Fdx (Table 4).
These results also indicate that the FNR, which is
encoded by TTC0096, transfers electrons from
NADPH to Fdx.
Characterization of the CYP175A1 system
reconstituted from its recombinant components
We attempted to reconstitute b-carotene hydroxylation

with other carotenoid oxygenases, such as carotenoid
dioxygenases, because detergents and phospholipids
presumably aid the solubilization of carotenoid and
thus increase its ability to access the active site of
carotenoid oxygenases [21–23]. Thus, we assessed the
effect of Tween 20 on b-carotene hydroxylation acti-
vity (Fig. 5C). Tween 20 stimulated b-carotene hydrox-
ylation activity, with maximal activity at 0.6–0.8%.
The turnover rate of the reconstitution system under
the optimal conditions was 12.4 nmol b-cryptoxan-
thinÆmin
)1
Ænmol
)1
CYP175A1. Furthermore, the K
m
and V
max
values for b-carotene hydroxylation by the
reconstitution system were determined under the opti-
mized conditions (Fig. 5D). The reaction followed
Michaelis–Menten kinetics, and the K
m
and V
max
values were 14.3 ± 1.6 lm and 18.3 ± 0.6 nmol
b-cryptoxanthinÆmin
)1
Ænmol
)1

(TTC1809) and FNR (TTC0096). Thus, the electron
transport system for CYP175A1 belongs to class I,
along with electron transport systems for other bacte-
rial cytochrome P450s, and is very different from
another thermophilic cytochrome P450 (CYP119)
system. In the CYP119 system from the thermophilic
archaeon S. solfataricus, electrons are transferred from
pyruvate via OFOR and Fdx to CYP119 [13,14]. Inter-
estingly, the electron transport system for CYP175A1
did not utilize OFOR, although the T. thermophilus
HB27 genome contains the genes encoding OFOR
(TTC1591 and TTC1592) [24]. An Fdx that contains
seven irons (one [4Fe–4S] cluster and one [3Fe–4S]
cluster) was discovered more than 20 years ago in
T. thermophilus [19], but its function has remained
unclear. Thus, this is the first report to demonstrate
that a protein encoded by TTC0096 functions as an
FNR in T. thermophilus, and that the seven-iron Fdx
functions as a redox partner of CYP175A1. Further-
more, we attempted to purify native CYP175A1, and
measured reduced CO difference spectra in order to
investigate whether or not CYP175A1 would be
expressed under the culture conditions used in this
study, but we could not purify native CYP175A1 and
detect an absorption peak at 450 nm (data not shown).
Nonetheless, very low b-carotene hydroxylation acti-
vity was detected in the presence of Fdx, FNR,
NADPH, and the cytosol of T. thermophilus (data not
shown), suggesting that CYP175A1 was expressed at
very low levels under the culture conditions used in

62
47.5
32.5
25
16.5
kDa
12 3 4 5
Wavelength (nm)
0.0
0.1
0.2
0.3
0.4
300 400 500 600
Absorbance
Absorbance
Wavelength (nm)
450
0.00
0.02
0.04
550350
Fig. 2. Purification and characterization of FNR from T. thermophi-
lus HB27. (A) SDS ⁄ PAGE of fractions containing FNR at each step
of purification. SDS ⁄ PAGE was carried out on a 15% polyacryl-
amide gel. Lane 1: molecular mass markers. Lane 2: cytosol of
T. thermophilus HB27 (20 lg). Lane 3: 100 m
M KCl fraction from a
DE52 column (14 lg). Lane 4: fraction eluted from a 2¢,5¢-ADP–
Sepharose column (4.6 lg). Lane 5: fraction eluted from a Mono Q

temperature. This was about 54-fold greater than the
turnover rate (0.23 nmol b-cryptoxanthinÆmin
)1
Ænmol
)1
CYP175A1) reported by Momoi et al. [11], who car-
ried out reconstitution using an artificial electron
transport system, putidaredoxin and putidaredoxin
reductase from the mesophilic bacterium P. putida.
Although CYP97A4 from Oryza sativa also catalyzes
the hydroxylation of b-carotene at the 3-position and
3¢-position in E. coli [28], the activity of CYP97A4 had
not been characterized in vitro. Thus, this is the first
report to characterize a cytochrome P450-type b-caro-
tene hydroxylase with its native electron transport
system.
In this study, the turnover rate of b-carotene
hydroxylation by the reconstitution system containing
CYP175A1, Fdx and FNR was about 5000-fold lower
than that of ferricyanide reduction by the FNR. The
reason for this discrepancy is unclear, but general
class I systems such as mitochondrial cytochrome P450
systems also show a turnover rate of substrates of
cytochrome P450 that is much lower than the turnover
rate of ferricyanide reduction by FNR [29–31].
As noted above, the CYP175A1 system produces
thermozeaxanthins and thermobiszeaxanthins for rein-
forcement of the cell membrane at high temperature
[16]. Most enzymes, including CYP175A1, that are
related to the carotenoid biosynthetic pathway are

)1
) Purification (fold) Yield (%)
Crude extract 474.5 118.3 0.2 1 100
DE52 51.0 85.4 1.7 7 72
ADP–Sepharose 1.5 69.6 46.2 185 59
Mono Q 0.4 29.9 77.8 312 25
Table 3. Kinetic parameters for the ferricyanide reduction activity
of FNR. Ferricyanide reduction activities were measured in 50 m
M
potassium phosphate buffer (pH 7.4) containing potassium ferri-
cyanide (1 m
M). The K
m
value for NADH was determined in the pre-
sence of FNR (200 n
M) and NADH (0.5–7.0 mM), and the K
m
value
for NADPH was determined in the presence of FNR (20 n
M) and
NADPH (2–100 l
M).
NADH NADPH
K
m
(lM) 2440 ± 546 4.1 ± 0.2
V
max
(nmolÆmin
)1

ronments.
Experimental procedures
Materials
T. thermophilus HB27 was a gift from S. Kuramitsu
(Department of Biology, Graduate School of Science,
Osaka University, Osaka, Japan). KOD Plus DNA poly-
merase was purchased from Toyobo (Osaka, Japan). Emul-
gen 911 was a gift from Kao Chemical (Tokyo, Japan).
NADPH, NADH and NADP
+
were purchased from
Oriental Yeast (Tokyo, Japan). a-Cyano-4-hydroxycinnamic
acid was obtained from Bruker Daltonics GmbH (Bremen,
Germany). Molecular mass standards for gel filtration
(MW-GF-200), glucose 6-phosphate and cytochrome c were
purchased from Sigma Chemical Co. (St Louis, MO, USA).
b-Carotene, glucose-6-phosphate dehydrogenase from yeast,
potassium ferricyanide, chloramphenicol, ampicillin, isopro-
pyl-thio-b-d-galactoside (IPTG) and phenylmethanesulfonyl
fluoride were obtained from Wako Pure Chemical indus-
tries (Osaka, Japan). Tween 20 was purchased from
Bio-Rad Laboratories (Hercules, CA, USA).
Cloning, expression and purification of CYP175A1
T. thermophilus HB27 was cultured at 70 °CinThermus
medium (4 g of tryptone, 2 g of yeast extract and 1 g of
NaCl per liter, pH 7.5). T. thermophilus HB27 genomic
DNA was extracted using the Wizard Genomic DNA Puri-
fication Kit (Promega, Madison, WI, USA). CYP175A1
(locus in the genome, TT_P0059) was amplified by PCR
using genomic DNA as a template and two oligonucleotide

pH
4 5 6 7 8
20
40
60
A
B
0
Residual activity (%)
100
80
60
40
20
0
Temperature (°C)
40 60 80 100
Fig. 3. Characterization of FNR. (A) Effect of pH on the activity of
FNR. The buffers used in this experiment were 50 m
M potassium
acetate buffer of pH range 4.0–6.0 (closed circles and solid line)
and 50 m
M potassium phosphate buffer of pH range 6.0–8.0 (open
circles and dotted line). Ferricyanide reduction assays were
performed in each buffer containing 1 m
M potassium ferricyanide,
FNR (30 n
M) and 1 mM NADPH at 50 °C. The values represent the
mean ± standard deviation (SD) of triplicate experiments. (B) Ther-
mostability of FNR. FNR (60 n

(Pharmacia). After the column had been washed with
50 mm potassium phosphate buffer (pH 6.3) containing
10% glycerol and 100 mm KCl, CYP175A1 was eluted with
a linear gradient of 100–600 mm KCl in 50 mm potassium
phosphate buffer (pH 6.3) containing 10% glycerol, at a
flow rate of 1.0 mLÆmin
)1
. Fractions exhibiting a ratio of
absorbance at 418 ⁄ 280 nm above 1.3 were pooled, dialyzed
against buffer A, and stored at )80 °C until use. The con-
centration of purified CYP175A1 was determined with an
extinction coefficient of 104 mm
)1
Æcm
)1
at 418 nm [4].
Approximately 5 mg of purified CYP175A1 was obtained
per 1 L of culture, and a single band was observed on
SDS ⁄ PAGE.
Purification of an electron transport system for
CYP175A1 from T. thermophilus HB27
T. thermophilus HB27 was cultured in Thermus medium
(total volume: 6 L) at 70 °C overnight. T. thermophilus
HB27 was harvested by centrifugation at 5000 g for
20 min. All purification steps were performed at room tem-
perature. The pellet was suspended in buffer B (20 mm
potassium phosphate buffer, pH 7.7, and 10% glycerol)
containing 1 mm phenylmethanesulfonyl fluoride and
0.1 mm EDTA, and the cell suspension was disrupted by
sonication. The cell debris was removed by centrifugation

EDTA), and the dialyzed solution was then loaded onto a
2¢,5¢-ADP–Sepharose column (Amersham Biosciences)
equilibrated with buffer C. After the column had been
washed with buffer C containing 150 mm KCl, the proteins
were eluted with buffer C containing 150 mm KCl and
1mm NADP
+
. The fraction eluted from the 2¢,5¢-ADP–
Sepharose column was dialyzed against buffer B. The
β-carotene
β-cryptoxanthin
Retention time (min)
10 20 300
A
454
0
20
40
60
80
100
A
B
Zeaxanthin
NADPH
NADP
+
e
–
FNR

a
Data from this study.
b
Data from Griffin et al. [32].
c
Data from
Yano et al. [4].
T. Mandai et al. Thermostable electron transport system
FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2423
dialyzed solution was loaded onto a Mono Q HR5 ⁄ 5
column equilibrated with buffer B, and the column was
washed with buffer B containing 50 mm KCl. The proteins
were eluted with a linear gradient of 50–200 mm KCl in
buffer B at a flow rate of 1.0 mLÆmin
)1
. The purified pro-
tein was dialyzed against buffer A and stored at )80 °C.
Identification of the purified electron transport
proteins
The electron transport protein purified from the 300 mm KCl
fraction eluted from the DE52 column was identified by
determining the N-terminal amino acid sequence of the puri-
fied protein, which was analyzed by an automated amino
acid sequencer (PPSQ-21A; Shimadzu, Kyoto, Japan),
according to the manufacturer’s instructions. The electron
transport protein purified from the 100 mm KCl fraction
eluted from the DE52 column was identified by MALDI-
TOF-MS. The purified protein was electrophoresed with
an SDS ⁄ polyacrylamide gel, and stained with Coomassie
Brilliant Blue R-250. The band containing the purified

Tween 20 (%)
0.0
0
5
10
15
0.5 1.0 1.5 2.0
Turnover rate
(nmol·min
–1
·nmol of CYP175A1
–1
)
Turnover rate
(nmol·min
–1
·nmol of CYP175A1
–1
)
Fdx (n
M
)
0
300 600 900 1200
0
2
4
6
8
β-carotene (µ

out as described in Experimental procedures, and the values represent the mean ± SD of triplicate experiments.
Thermostable electron transport system T. Mandai et al.
2424 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS
T. thermophilus 1 MAADHTDVLIVGAGPAGLFAGFYVGMRGLSFRFVDPLPEPGGQLTAL 47
E.
coli 1 MGTTKHSKLLILGSGPAGYTAAVYAARANLQPVLITGM-EKGGQLTTT 47
A.
pernix 1 MPLRLSAVRAPKIPRGEEYDTVIVGAGPAGLSAAIYTTRF-LMSTLIVSM-DVGGQLNLT 58
. :*:*:****
* *. * :: : : ****.
1
T.
thermophilus 48 YPEKYIYDVAG-FPKVYAKDLVKGLVEQVAPFNPVYSLGERAETLE-REGDLFKVTTSQG 105
E.
coli 48 T EVENWPGDPNDLTGPLLMERMHEHATKFETEIIFD-HINKVD-LQNRPFRLNGDNG 102
A.
pernix 59 N WIDDYPG-MGGLEASKLVESFKSHAEMFGAKIVTGVQVKTVDRLDDGWFLVRGSRG 114
: : .* : . *:: : .:. * . . : :.:: :. * : *
T.
thermophilus 106 NAYTAKAVIIAAGVGAFEPRRIGAPGEREFEGRGVYYAVKSKA-EFQGK-RVLIVGGGDS 163
E.
coli 103 -EYTCDALIIATGASA RYLGLPSEEAFKGRGVSACATCDG-FFYRNQKVAVIGGGNT 157
A.
pernix 115 LEVKARTVILAVGSRR RKLGVPGEAELAGRGVSYCSVCDAPLFKGKDAVVVVGGGDS 171
::*:*.* * :* *.* : **** . .
* : * ::***::
2 3
T.
thermophilus 164 AVDWALNLLDTARRITLIHRRPQFRAHEASVKELMKAHEEGRLEVLTPYELRRVEGDER- 222
E.

Nostoc sp. PCC 7120 FNR
Z. mays FNR
S. oleracea FNR
E .coli FNR
R. capsulatus FNR
A. vinelandii FNR
Plant-type FNRs
Plastidic-type
Bacterial-type
New type
A
B
ADR-like
ONFR-like
GR-type FNRs
Fig. 6. (A) Multiple alignment of the amino acid sequences of FNR from T. thermophilus HB27, TR from E. coli, and TR from A. pernix.
Accession numbers (NCBI) are: FNR from T. thermophilus HB27, YP_004071; TR from E. coli, NP_415408; and TR from A. pernix,
NP_147693. Asterisks indicate identical amino acid residues. Colons indicate conservative replacements, and single dots indicate less
conservative replacements. Underlines 1, 2 and 3 indicate the FAD-binding site, the redox-active site, and the NADPH-binding site,
respectively. (B) Phylogenetic tree of FNR from different sources. The phylogenetic tree was constructed using the program
CLUSTALW
( The accession numbers are: FNR from Spinacia oleracea, AAA34029; FNR from Nostoc sp. PCC 7120, NP_488161;
FNR from Zea mays, NP_001105568; FNR from E. coli, NP_418359); FNR from Azotobacter vinelandii, ZP_00417949; FNR from Rhodobact-
er capsulatus, AAF35905; ADR from Homo sapiens, AAB59498; adrenodoxin reductase from Saccharomyces cerevisiae, AAB64812; FprA
from Mycobacterium tuberculosis, O05783; BphA4 from Pseudomonas sp. KKS102, BAA04112; putidaredoxin reductase from P. putida,
AAA25758; FNR from M. tuberculosis H37Rv, NP_215202; YumC from B. subtilis, CAB15201; and FNR from C. tepidum, NP_662397.
T. Mandai et al. Thermostable electron transport system
FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2425
by MALDI-TOF-MS (Ultraflex; Bruker Daltonics GmbH).
Protein identification was carried out by a search of the data-

29.0 mm
)1
Æcm
)1
at 408 nm [13].
Cloning, expression and purification of FNR
FNR (TTC0096) was amplified by PCR using geno-
mic DNA as a template and two oligonucleotide prim-
ers, 5¢-GGAATTCCATATGGCGGCGGAC CACACGGA
CGT-3¢ (forward primer) and 5¢-CGCGGATCCTAGG
TCCCGGGGGCGGCCTTCTC-3¢ (reverse primer). The
PCR product was inserted into the pET-21a vector, and the
construct was designated pET–FNR. E. coli BL21(DE3)
Codon Plus cells were transformed with pET–FNR. The
transformant was grown in 2 · YT medium containing
chloramphenicol and ampicillin at 37 °CuptoanD
600
of
1.0, and FNR expression was induced by treatment with
1.0 mm IPTG for 5 h at 37 °C. Cells were harvested by cen-
trifugation at 5000 g for 20 min, and the pellet was sus-
pended in buffer B containing 1 mm phenylmethanesulfonyl
fluoride and 0.1 mm EDTA. The crude extract of E. coli
was prepared as described above. The extract was incu-
bated at 70 °C for 30 min, and then centrifuged at 20 000 g
for 30 min at 4 °C. The heat-treated supernatant was puri-
fied with a DE52 column, a 2¢,5¢-ADP–Sepharose column
and a Mono Q column under the conditions described
above. The purified FNR was dialyzed against buffer A,
and stored at )80 °C. The concentration of the purified

containing 2.3% Tween 20 (v ⁄ v), and then mixed
vigorously and vacuum-dried. The resulting residue was
dissolved in 99 lL of the reaction buffer (200 lm b-caro-
tene solution). All reactions were carried out in 2 mL
tubes with caps.
To purify the electron transport system for CYP175A1,
b-carotene hydroxylation reactions with CYP175A1
(0.5 lm) and the electron transport system were per-
formed in buffer A containing 20 lm b-carotene (total
volume, 200 lL). The reaction mixtures were incubated at
65 °C for 3 min, and the reactions were initiated by the
addition of 2 lL of 100 mm NADPH. After 2 min at
65 °C, ice-cold acetonitrile ⁄ chloroform [4 : 1 (v ⁄ v),
1.0 mL] was added to extract the reaction products. The
tubes were placed on ice for 5 min, and then centrifuged
at 13 000 g for 10 min. The supernatant was directly ana-
lyzed by RP-HPLC. The HPLC analysis was performed
using an HPLC system (Prominence; Shimadzu, Kyoto,
Japan) equipped with an ODS-100S column (150 ·
4.6 mm; Tosoh, Tokyo, Japan), and acetonitrile ⁄ metha-
nol ⁄ isopropanol (85 : 10 : 5) was used as the mobile
phase, at a flow rate of 1 mLÆmin
)1
. To determine the
optimal reaction conditions, we assessed the effects of pH
(4.0–7.4), Fdx (30–960 nm) and Tween 20 (0.1–1.6%) on
b-carotene hydroxylation activity. The b-carotene hydrox-
ylation reactions were carried out under the conditions
described above, and the products were extracted with
ice-cold acetonitrile (1.0 mL). For the kinetic analysis,

flow, 5 LÆmin
)1
;N
2
gas temperature, 250 °C.
Ferricyanide and cytochrome c reduction assay
Unless otherwise stated, ferricyanide reduction assays
were performed in 50 mm potassium phosphate buffer
(pH 7.4) containing potassium ferricyanide (1 mm)at
25 °C (total volume, 500 lL). For the kinetic analysis,
the concentration of NADPH was kept constant by
regeneration with glucose 6-phosphate and glucose-6-
phosphate dehydrogenase from yeast. Cytochrome c
reduction assays were performed in 50 mm potassium
phosphate buffer (pH 7.4) containing horse heart cyto-
chrome c (0.1 mm), NADPH (0.5 mm), FNR (50 nm)
and Fdx (50–500 nm)at50°C (total volume,
500 lL). Ferricyanide reduction activity was calculated
from the decrease in absorbance at 420 nm (e
420 nm
=
1.02 mm
)1
Æcm
)1
). Cytochrome c reduction activity was
calculated from the increase in absorbance at 550 nm
(e
550 nm
= 21.0 mm

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