Báo cáo khoa học: The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXa reductase from the cyanobacterium Synechocystis PCC6803 pptx

The effect of pH on the initial rate kinetics of the dimeric
biliverdin-IXa reductase from the cyanobacterium
Synechocystis PCC6803
Jerrard M. Hayes and Timothy J. Mantle
School of Biochemistry and Immunology, Trinity College, Dublin, Ireland
Introduction
Cyanobacteria utilize linear tetrapyrroles as light-har-
vesting pigments that are found covalently attached to
phycobiliproteins in the ‘light pipes’ known as phyco-
bilisomes [1]. Two genera, Synechococcus and Prochlo-
rococcus, are suggested to be responsible for 25% of
global photosynthesis [2] and, although some strains of
Prochlorococcus do not express phycobiliproteins (e.g.
MED4), others (e.g. SS120) express a phycourobilin-
containing type-III phycoerythrin [3]. Linear tetrapyr-
role metabolism in cyanobacteria is therefore a major
physiological pathway. Cyanobacteria express ferre-
doxin-dependent bilin reductases (PcyA, PebA and
PebB) that synthesize phycocyanobilin and phycoery-
throbilin from biliverdin-IXa [4]. These linear tetrapyr-
Keywords
biliverdin reductase; compulsory ordered
mechanism; dimer; pH; Synechocystis
Correspondence
J. M. Hayes, School of Biochemistry and
Immunology, Trinity College, Dublin 2,
Ireland
Fax: +353 677 2400
Tel: +353 895 1612
E-mail:
(Received 23 April 2009, revised 9 June

2009, accepted 11 June 2009)
doi:10.1111/j.1742-4658.2009.07149.x
Biliverdin-IXa reductase from Synechocystis PCC6803 (sBVR-A) is a stable
dimer and this behaviour is observed under a range of conditions. This is in
contrast to all other forms of BVR-A, which have been reported to behave
as monomers, and places sBVR-A in the dihydrodiol dehydrogenase ⁄ N-ter-
minally truncated glucose–fructose oxidoreductase structural family of
dimers. The cyanobacterial enzyme obeys an ordered steady-state kinetic
mechanism at pH 5, with NADPH being the first to bind and NADP
+
the
last to dissociate. An analysis of the effect of pH on k
cat
with NADPH as
cofactor reveals a pK of 5.4 that must be protonated for effective catalysis.
Analysis of the effect of pH on k
cat
⁄ K
m
NADPH
identifies pK values of 5.1
and 6.1 in the free enzyme. Similar pK values are identified for biliverdin
binding to the enzyme–NADPH complex. The lower pK values in the free
enzyme (pK 5.1) and enzyme–NADPH complex (pK 4.9) are not evident
when NADH is the cofactor, suggesting that this ionizable group may inter-
act with the 2¢-phosphate of NADPH. His84 is implicated as a crucial resi-
due for sBVR-A activity because the H84A mutant has less than 1% of the
activity of the wild-type and exhibits small but significant changes in the
protein CD spectrum. Binding of biliverdin to sBVR-A is conveniently
monitored by following the induced CD spectrum for biliverdin. Binding of

biliverdin to wild-type sBVR-A induces a P-type spectrum. The H84A
mutant shows evidence for weak binding of biliverdin and appears to bind a
variant of the P-configuration. Intriguingly, the Y102A mutant, which is
catalytically active, binds biliverdin in the M-configuration.
Abbreviations
hBVR-B, human biliverdin-IXb reductase; HSA, human serum albumin; sBVR-A, biliverdin-IXa reductase from Synechocystis PCC6803; GST,
glutathione S-transferase.
4414 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS
roles are then incorporated into the phycobilisome
complex. Intriguingly, some strains of cyanobacteria
express biliverdin-IXa reductase (BVR-A), which catal-
yses the pyridine nucleotide-dependent reduction of
biliverdin-IXa to bilirubin-IXa. The first report of a
cyanobacterial BVR-A was from Synechocystis
PCC6803 (sBVR-A) [5] and BVR-A-like sequences are
also clearly identifiable in Gleobacter, Anabena, Nostoc
and Trichodesmium (E. Franklin & T. J. Mantle,
unpublished results). Schluchter and Glazer [5]
reported on the unusual acidic pH optimum for sBVR-
A. They also describe features of a Synechocystis
PCC6803 strain lacking sBVR-A, which they interpret
as indicating that the reaction product, bilirubin-Ixa,
plays a role in phycobiliprotein biosynthesis [5]. We
have been intrigued that sBVR-A can potentially divert
flux from phycobilin biosynthesis and also potentially
reduce the phycobilins to the corresponding rubin, a
reaction clearly catalysed in vitro [5], albeit with a rela-
tively high K
m
for phycocyanobilin [5].

Questions on the possible function of BVR-A in
cyanobacteria parallel a major re-evaluation of the
function of BVR-A in mammals. Once considered to
play a role solely in the elimination of excess haem, it
is now implicated in the maintenance of a major anti-
oxidant, bilirubin-IXa [6]. At high doses, biliverdin
appears to tolerize the immune system of recipients
undergoing organ transplantation in animal studies
[7,8], although it is presently unclear whether this
effect is caused by biliverdin-IXa or bilirubin-IXa.
Because BVR-A is reponsible for the production of bil-
irubin-IXa in neonates at birth, it is also a pharmaco-
logical target for treating neonatal jaundice [9]. The
rat [10,11] and human enzymes [12] have been crystal-
lized; however, although there are complexes with
NAD
+
[11] and NADP
+
[12], little is known about
the biliverdin binding site. Although the mammalian
enzymes have received the most attention, comparative
studies on the salmon and Xenopus tropicalis enzymes
are available [13,14]. In this respect, the enzyme from
Synechocystis is of considerable interest because it
exhibits a narrow acidic pH optimum compared to the
broad range of pH that can support activity for the
mammalian enzymes [5,14]. The cyanobacterial enzyme
is also refractory to activation by inorganic phosphate
when NADH is the cofactor [14]. In preliminary exper-

iments, we observed that sBVR-A is not subject to the
potent substrate inhibition observed with the mamma-
lian enzymes and is therefore the first candidate,
among all BVR-A forms studied to date, where a com-
plete initial rate study can be completed in the absence
of a biliverdin-binding protein as well as at the opti-
mum, presumably physiological, pH. In preliminary
gel filtration experiments, we have also shown that the
Synechocystis enzyme behaves as a dimer and such
studies are extended to include the light-scattering and
analytical ultracentrifugation studies described here.
We report a complete initial rate study, including the
effect of pH on the kinetic parameters and site-directed
mutagenesis studies, to gain an understanding of the
function of sBVR-A in cyanobacteria and also to
increase our knowledge of the mechanism of an
enzyme closely related to a pharmacological target for
neonatal jaundice.
Results
The expression vector pETBVR-A allowed us to rou-
tinely prepare 20 mg of electrophoretically homoge-
nous sBVR-A from 4 L of culture using Escherichia
coli BL21 (DE3) cells. Using this approach, the
enzyme has two additional residues at the N-terminus
(Ser-Gly) but lacks the His-tag in the preparation
reported earlier [5]. The enzyme was colourless; how-
ever, the UV spectrum revealed significant absorbance
at 260 nm. Analysis of the protein sample by HPLC
revealed that, in addition to the protein, there was one
major and two minor peaks that absorbed at 254 nm.

The major peak, which eluted at 38 min, was identified
as NADPH by its retention time, fluorescence emission
spectrum and UV absorbance spectrum. We have not
pursued the identity of the two minor peaks. All three
compounds were released from the enzyme when it
was bound to 2¢,5¢-ADP-sepharose and, under these
conditions, the enzyme was eluted without contamina-
tion. In preliminary experiments, we observed that
sBVR-A eluted just before BSA on gel filtration in
25 mm sodium citrate pH 5 (the optimum pH for
activity; see below) and, by comparison with the elu-
tion volume of standard proteins, this is consistent
with a molecular mass of 69 kDa at 20 ° C and 74 kDa
at 4 °C (Table 1). Although the enzyme is less active
at pH 7.5, gel filtration was carried out at this pH and
at 20 °C as well as 4 °C and, under all these condi-
tions, the molecular mass of the enzyme corresponds
to that of the dimer (Table 1). This result is novel
because all BVR-As described to date have been
reported to behave as monomers [15–17]. To confirm
that sBVR-A is a dimer, we examined the native
molecular mass using light-scattering and analytical
ultracentrifugation (both sedimentation velocity and
sedimentation equilibrium) and the results obtained
are provided in Table 1. These confirm the results of
the gel filtration and are consistent with the dimeric
nature of sBVR-A because several purified prepara-
tions have been shown to run with a subunit molecular
J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A
FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4415

mass of 34 kDa on SDS ⁄ PAGE. Prior to a detailed
kinetic analysis, the stability of sBVR-A at a range of
pH values was determined. Over the pH range 5–7, the
enzyme did not lose any activity when pre-incubated
for 180 min. The enzyme was unstable outside this
range, being particularly unstable below pH 4.5. At
pH 4, the half life was 30 s. At pH 8, the enzyme
started to lose activity after 60 min and, at pH 9,
retained 75% of the activity after 60 min of incuba-
tion. The initial rate demonstrates a linear dependence
on enzyme concentration (from 0.5–5 lg ⁄ mL) when
assayed at pH 5 with NADPH or NADH as cofactor.
All initial rate experiments were conducted within this
range of enzyme concentration. In a preliminary set of
experiments, there was no substrate inhibition up to a
biliverdin concentration of 50 lm, in clear distinction
to the mammalian enzymes.
Initial rate experiments with NADPH or NADH as
the variable substrate were carried out by working at
various fixed concentrations of biliverdin-IXa and
varying the concentration of NADPH from 5–100 lm
or NADH from 50–1000 lm. The ‘fixed’ concentra-
tions of biliverdin were also varied from 0.5–10 lm to
yield the plot for NADPH as the variable substrate
shown in Fig. 1A. The apparent V
max
values for
NADPH as the variable substrate were then replotted
against the biliverdin concentration (Fig. 1B) to yield
the true V

max
and true K
m
values for biliverdin with
NADPH as cofactor (Table 2). The initial rate mea-
surements also yielded linear double-reciprocal plots
(not shown) that intersected to the left of the recipro-
cal initial rate axis, suggesting that the mechanism was
sequential. However, these experiments could not iden-
tify which of the substrates bound first or whether
there was any particular order in their binding. A simi-
lar pattern was obtained with NADH as the variable
substrate (data not shown).
Initial rate experiments with biliverdin-IXa as the
variable substrate were carried out similarly to those
described for NADPH but using various ‘fixed’ con-
centrations of NADPH and varying the biliverdin-IXa
concentration in the range 0.5–10 lm. The data were
Table 1. Relative molecular mass of native sBVR-A. AUC, area
under the curve.
pH
Temperature
(°C)
MW
(kDa)
Gel filtration 5 20 69
54 74
7.5 20 66
7.5 4 64
Light scattering 5 20 73.2

7.5 20 66.2
AUC velocity 5 11 71
5 21 80.1
7.5 11 80.4
7.5 21 80
AUC equilibrium
11 612 g 5 4 69.2
18 144 g 5 4 63.9
26 127 g 54 55
11 612 g 7 4 73.8
18 144 g 7 4 77.7
26 127 g 74 72
A
B
Fig. 1. Initial rate kinetics of sBVR-A with NADPH as the variable
substrate. (A) The reaction was conducted in 100 m
M sodium cit-
rate buffer (pH 5) and the reaction was initiated by the addition of
sBVR-A (5 lg). The concentrations of NADPH are indicated and the
concentrations of biliverdin-IXa were 0.5 l
M ( ), 1 lM ( ), 2 lM
(.), 5 lM (r) and 10 lM (•). Each point represents the mean and
the error bars represent the standard deviation of triplicate values.
The curves are least squares fits to a rectangular hyperbola. (B) A
replot of the apparent V
max
from (A) against the concentrations of
biliverdin-IXa. The curve is a least squares fit to a rectangular hyper-
bola and the error bars are the standard errors from the fits in (A).
Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle

4416 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS
fitted to a rectangular hyperbola (Fig. 2A). The true
V
max
and K
m
for NADPH were calculated by replot-
ting the apparent V
max
values (obtained from the fits
in Fig. 2A) against the NADPH concentration
(Fig. 2B) and the kinetic constants are shown in
Table 2. The initial rate data with biliverdin-IXa as
the variable substrate also yielded linear intersecting
double-reciprocal plots that were consistent with a
sequential mechanism (data not shown). Although
these data sets indicate that the enzyme obeys a
sequential mechanism, product inhibition patterns are
required to distinguish between steady-state ordered,
random sequential and Theorell–Chance mechanisms.
NADP
+
inhibition with NADPH as the variable
substrate was carried out at saturating (10 lm) levels
of biliverdin. The inhibitory concentrations of NADP
+
were in the range 0–100 lm, whereas the concentration
of NADPH varied in the range 5–100 lm. Curves were
again fitted to the initial rate data set and used to yield
double-reciprocal plots (Fig. 3A). The pattern of the

double-reciprocal plots shows that NADP
+
exhibits
competitive kinetics against NADPH. When the slope
values of the double-reciprocal plots were replotted
against the inhibitor concentration, a linear relation-
ship was obtained (Fig. 3B) and was used to determine
the inhibitor constant K
is
for NADP
+
, which is shown
in Table 3. NADP
+
inhibition was also carried out
with biliverdin as the variable substrate. These experi-
ments were performed as described for NADPH but
keeping the NADPH concentration constant at nonsat-
urating (10 lm) and saturating (1 mm) levels of
NADPH and varying the biliverdin concentration in
the range 0.5–10 lm. A concentration of 1 mm was
used for NADPH to ensure saturation. NADP
+
showed mixed inhibition against biliverdin at nonsatu-
rating levels of NADPH. When the experiment was
repeated at saturating levels of NADPH, no inhibition
A
B
Fig. 2. Initial rate kinetics of sBVR-A with biliverdin-IXa as the vari-
able substrate. (A) The reaction was conducted in 100 m

M sodium
citrate buffer (pH 5) and the reaction was initiated by the addition of
sBVR-A (5 lg). The concentrations of biliverdin-IXa are indicated and
the concentrations of NADPH were 5 l
M ( ), 10 lM ( ), 20 lM (.),
50 l
M (r) and 100 lM (•). Each point represents the mean and the
error bars represent the standard deviation of triplicate values. The
curves are least squares fits to a rectangular hyperbola. (B) A replot
of the apparent V
max
from (A) against the concentrations of NADPH.
The curve is a least squares fit to a rectangular hyperbola and the
error bars are the standard errors from the fits in (A).
Table 2. Kinetic parameters for wild-type and mutant forms of
sBVR-A.
sBVR-A
Variable
substrate
V
max
(lmolÆmin
)1
Æ
mg
)1
)
K
m
(lM)

k
cat
(s
)1
)
Wild-type NADPH 0.78 ± 0.06 10.78 ± 3.2 0.44 ± 0.034
Biliverdin 0.79 ± 0.07 2.32 ± 0.59 0.45 ± 0.02
NADH 0.29 ± 0.04 207 ± 66 0.17 ± 0.023
Biliverdin 0.24 ± 0.027 1.6 ± 0.55 0.15 ± 0.015
Y102A NADPH 0.33 ± 0.06 3.55 ± 3.2 0.18 ± 0.034
Biliverdin 0.33 ± 0.022 16.19 ± 1.62 0.18 ± 0.012
R185A NADPH 0.089 ± 0.006 4.54 ± 1.4 0.05 ± 0.034
Biliverdin 0.089 ± 0.01 3.22 ± 0.9 0.05 ± 0.006
H129A NADPH 0.68 ± 0.01 4.53 ± 0.32 0.39 ± 0.006
Biliverdin 0.68 ± 0.055 1.3 ± 0.34 0.39 ± 0.03
H126A NADPH 0.62 ± 0.1 23.3 ± 10 0.35 ± 0.06
Biliverdin 0.64 ± 0.05 4.67 ± 0.72 0.36 ± 0.03
H97A NADPH 0.58 ± 0.05 5.81 ± 2.3 0.33 ± 0.03
Biliverdin 0.58 ± 0.026 2.26 ± 0.28 0.33 ± 0.015
H84A NADPH
Biliverdin
0.008
0.008
Unable to
calculate
E101A NADPH 0.27 ± 0.034 8.8 ± 4 0.15 ± 0.02
Biliverdin 0.25 ± 0.22 21.66 ± 24.55 0.14 ± 0.13
D285A NADPH 0.1 ± 0.006 1 ± 0.73 0.057 ± 0.01
Biliverdin 0.11 ± 0.013 1.60 ± 0.6 0.062 ± 0.007
J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A

FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4417
was observed (data not shown). The inhibition con-
stants (K
is
from the slope replot and K
ii
from the inter-
cept replot) for NADP
+
with biliverdin as the variable
substrate are shown in Table 3. NAD
+
showed com-
petitive kinetics against NADH and was a mixed
inhibitor against biliverdin at nonsaturating (100 lm)
levels of NADH (Table 3).
Bilirubin inhibition with biliverdin as the variable
substrate was conducted at saturating (100 lm) levels
of NADPH and revealed that bilirubin is a mixed
inhibitor against biliverdin at saturating levels of
NADPH. Product inhibition experiments with bilirubin
as an inhibitor were also conducted with NADPH as
the variable substrate (5–100 lm) at nonsaturating
(1 lm) and saturating (10 lm) levels of biliverdin. Ini-
tial rate data for nonsaturating levels of biliverdin
show that bilirubin exhibits mixed inhibition kinetics
at nonsaturating levels of biliverdin and, when the
experiment was repeated at saturating levels of biliver-
din, the inhibition becomes uncompetitive (Fig. 4).
Bilirubin exhibits mixed inhibition against NADH at

nonsaturating levels of biliverdin and mixed inhibition
against biliverdin at saturating levels of NADH (data
not shown). Inhibition constants are shown in Table 3.
These product inhibition patterns are entirely consis-
tent with sBVR-A obeying a steady-state ordered
mechanism at pH 5, with NADPH being the first to
bind and NADP
+
the last to dissociate.
Inorganic phosphate anion has been shown to be an
activator of human BVR-A [14]. Increasing amounts
of sodium phosphate (0–100 mm) were added to the
sBVR-A assay using both NADH and NADPH as
cofactor and at pH 5 and pH 7. The pH was moni-
tored before and after the assay to ensure that it did
not change significantly when adding increasing
amounts of phosphate. The effect of ionic strength was
found to be minimal. Inorganic phosphate was found
to have no effect on sBVR-A activity with either cofac-
tor at either pH. This is a major discriminating feature
between the cyanobacterial enzyme and the vertebrate
BVR-A family members.
It is often the case that, when determining the effect
of pH on the kinetic parameters of a two-substrate
enzyme, one substrate is held at 10 · K
m
(91% saturat-
ing) and the variation of initial rate with the concen-
tration of the second substrate is then used to estimate
k

cat
and the K
m
for the variable substrate. However,
the assumption that a concentration that saturates at
one pH will saturate at all the pH values under investi-
gation is not without risk. All the initial rate parame-
ters reported in the present study were determined in
accordance with the classic analysis of Florini and Ves-
tling [18] to calculate K
m
and V
max
. The effect of pH
on k
cat
was investigated over the pH range 4.25–7.0
with both NADPH and NADH. The values measured
for k
cat
are shown when the data set is described with
NADPH or biliverdin as the variable substrate. The
same k
cat
profile should be obtained (irrespective of
which substrate is held as the variable) and this is
clearly seen in Fig. 5A. Evidently, there is a pK at 5.4
for the ‘less acidic’ limb of the pH curve defining a
side chain that must be protonated for catalysis to
occur. There is no co-operativity for this protonation

because the plot of log k
cat
versus pH gives a slope of
approximately –1 (Fig. 5B). On the ‘more acidic’ limb
A
B
Fig. 3. Product inhibition by NADP
+
with NADPH as the variable
substrate. The reaction was conducted in 100 m
M sodium citrate
buffer (pH 5) and the reaction was initiated by the addition of
sBVR-A (5 lg). Biliverdin-IXa was held constant (10 l
M) at saturat-
ing levels and the levels of NADPH are indicated. The concentra-
tions of NADP
+
were 0 lM ( ), 10 lM ( ), 20 lM (.), 50 lM (r)
and 100 l
M (•). (A) The data are represented as a double-reciprocal
plot and (B) a slope replot (apparent K
m
⁄ V
max
from fits to a rectan-
gular hyperbola) against the concentration of NADP
+
.
Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle
4418 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS

of this curve, there is a second pK (4.7) and proton-
ation of this group reduces the k
cat
(but only by 50%).
Great care has to be taken because the enzyme is
highly unstable at the ‘more acidic’ pH values. Initial
rates were obtained at pH 4 during the first few sec-
onds of the reaction under conditions when at least
90% of the activity was retained; however, these are
clearly not ideal conditions. With NADH as cofactor,
the pK on the ‘less acidic’ limb is clearly not co-opera-
tive (data not shown) and has a similar value (5.7) to
that observed with NADPH (5.4). It is intriguing that
the k
cat
on the ‘more acidic’ limb shows very little
dependence on pH with NADH as the cofactor.
The effect of pH on the k
cat
⁄ K
m
values for NADPH
and NADH was also analysed. The log plot for
k
cat
⁄ K
m
is shown for the NADPH (Fig. 6A) and bili-
verdin (Fig. 6B) data sets and for the NADH (Fig. 6C)
and biliverdin (Fig. 6D) data sets. With NADPH as

Table 3. Initial rate kinetic parameters for product inhibition studies of sBVR-A.
Inhibitor Variable substrate Fixed substrate Inhibition K
is
(lM) K
ii
(lM)
NADP
+
NADPH Biliverdin 10 lM (saturating) Competitive 12.7 –
NADP
+
Biliverdin NADPH 10 lM (nonsaturating) Mixed 26 51
NADPH 1000 l
M (saturating) No inhibition – –
NAD
+
NADH Biliverdin 10 lM (saturating) Competitive 613
NAD
+
Biliverdin NADH 100 lM (nonsaturating) Mixed 2615 1771
NADH 1000 l
M (saturating) No inhibition – –
Bilirubin NADPH Biliverdin 1 l
M (nonsaturating) Mixed 13 28
Biliverdin 10 l
M (saturating) Uncompetitive – 17.5
Bilirubin NADH Biliverdin 1 l
M (nonsaturating) Mixed 5.2 9.6
Bilirubin Biliverdin NADPH 100 l
M (saturating) Mixed 13 28

Bilirubin Biliverdin NADH 1000 l
M (saturating) Mixed 16 42
Fig. 4. Product inhibition by bilirubin-IXa with NADPH as the vari-
able substrate at saturating levels of biliverdin. (A) The reaction
was conducted in 100 m
M sodium citrate buffer (pH 5) and the
reaction was initiated by the addition of sBVR-A (5 lg). Biliverdin-
IXa was held constant at saturating levels (10 l
M) and the concen-
trations of NADPH are indicated. The concentrations of bilirubin-IXa
were 0 l
M ( ), 1 lM ( ), 2 lM (.), 5 lM (r) and 10 lM (•). The
data are represented as a double-reciprocal plot.
A
B
Fig. 5. Effect of pH on k
cat
with NADPH as cofactor: 25 mM
sodium citrate (pK values of 3.13, 4.76 and 6.4) was used as buffer
over the entire pH range studied. (A) Values for k
cat
were obtained
with NADPH as the variable substrate (
•) and with biliverdin-IXa as
the variable substrate (
). (B) The log ⁄ log plot for (A) is shown.
J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A
FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4419
cofactor, the k
cat

⁄ K
m
data reveal two pK values of 5.1
and 6.1 with NADPH as the variable substrate and 4.9
and 5.6 with biliverdin as the variable substrate. This
is consistent with two ionizing groups in the free
enzyme with pK values of 5.1 and 6.1 that define
NADPH binding. Interestingly, with NADH (Fig. 6C)
and biliverdin (Fig. 6D) as the variable substrates,
there is only a single pK (i.e. 5.3 for NADH binding
to the free enzyme and 5.5 for biliverdin binding to the
enzyme–NADH complex). The only difference between
NADPH and NADH is the 2¢-phosphate on NADPH
and it is tempting to suggest that there may be a disso-
ciable group with a pK of 5.1 in the free enzyme (4.9
in the enzyme–NADH complex) that is not involved in
binding NADH. This pK may be associated with an
ionizing residue that is involved in binding the 2¢-phos-
phate of NADPH.
The effect of pH on the initial rate kinetics is consis-
tent with two ionizing groups in the enzyme active site
involved in binding NADPH, which may be perturbed
slightly in the enzyme–NADPH complex but which are
both required for binding biliverdin. In the ternary com-
plex, a group with a pK of 5.4 must be protonated for
efficient catalysis with NADPH (in the case of the ter-
nary complex with NADH as cofactor, this pK is 5.7).
The nature of the second pK in the ternary complex
with NADPH (4.7) is unclear. There is no analogous
pK in the binding of NADH and it is not readily appar-

ent in the ternary complex with NADH as cofactor.
To identify the ionizing residues, we have attempted
to crystallize sBVR-A, so far without success. We have
therefore built a model using the rat enzyme as a tem-
plate and this is shown in Fig. 7. In this model, we
have highlighted residues from the sBVR-A model that
are candidates for the ionizing residues. These include
four His (84, 97, 126 and 129) one Glu (101), one Asp
(285) and one Tyr (102) residue. All were mutated to
Ala residues and the sequences confirmed. The gluta-
thione S-transferase (GST) fusions were purified, the
GST domain cleaved and removed by affinity chroma-
tography and the mutant sBVR-As analysed in terms
of CD spectra, induced CD spectra for biliverdin and
initial rate kinetic parameters. The kinetic parameters
of all the mutants are shown in Table 2. This clearly
rules out His97, His126 and His129, which have k
cat
and K
m
values that are very close to those displayed
by the wild-type enzyme. In addition. these three His
to Ala mutants show CD spectra and induced CD
spectra for biliverdin that are very close to those
exhibited by the wild-type enzyme (Fig. 8A). However,
a clear candidate for a key active site residue is His84.
The specific activity of the H84A mutant is 1% of the
wild-type and is so low that we were unable to
A
B

C
D
Fig. 6. log k
cat
⁄ K
m
versus pH for NADPH and NADH as the variable
substrates. (A) Log k
cat
⁄ K
m
with NADPH as the variable substrate.
(B) Log k
cat
⁄ K
m
with biliverdin as the variable substrate and NADPH
as cofactor. (C) Log k
cat
⁄ K
m
with NADH as cofactor. (D) Log k
cat
⁄ K
m
with biliverdin as the variable substrate and NADH as cofactor.
Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle
4420 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS
determine the kinetic parameters with confidence.
Examination of the CD spectrum of H84A protein

reveals that it is close to, but not identical with, that
of the native enzyme (data not shown). We suggest
that H84A may be the residue responsible for proton-
ating the pyrollic nitrogen prior to hydride transfer
(see Discussion); however, we cannot discount a mod-
est global structural change having some role in the
decreased catalytic activity. It should be noted that, in
this respect, the H84A mutant was isolated with bound
nucleotide and is clearly able to bind biliverdin-IXa
(Fig. 8B), suggesting that both substrates are able to
bind to the H84A mutant.
The binding of biliverdin to wild-type sBVR-A stabi-
lizes the helical P-configuration (Fig. 8A) of the linear
tetrapyrrole, also known as the ‘lock washer’ [19] and,
by this criteria, biliverdin can be seen to bind weakly
to the H84A mutant (Fig. 8B), albeit with a broaden-
ing of the positive ellipticity into a peak at 400 nm
and a significant shoulder at 325 nm. Two of the
mutants (R185A and D285A) have k
cat
values that are
only 10% of wild-type (Table 2). The D285A mutant
has modest changes in the K
m
values and the induced
CD spectrum for biliverdin is similar to wild-type. The
R185A mutant also shows similar K
m
values to the
wild-type; however, the positive ellipticity of the

induced CD for biliverdin shows a sharp peak at
400 nm (the wild-type shows a broad peak centred at
390 nm) and a clear minor peak at 325 nm (Fig. 8C).
In the case of the E101A mutant, there is a significant
increase in the K
m
for biliverdin (nine-fold) and the
k
cat
is reduced to a third of that of the wild-type
(Table 2). The induced CD spectrum for biliverdin
bound to the E101A mutant shows a considerably
reduced amplitude, with the positive ellipticity split
into two peaks at 325 nm and 400 nm (Fig. 8D). In
this case, the trough is centered at 580 nm (compared
to 700 nm in the wild-type). Intriguingly the Y102A
mutant exhibits CD behaviour that reflects the M-con-
figuration (Fig. 8E). The ability of this mutant to
stabilize the opposite enantiomer is associated with a
modest (50%) drop in the k
cat
and a seven-fold
increase in the K
m
for biliverdin.
Discussion
All mammalian forms of BVR-A are reported to
behave as monomers. These include the enzymes from
pig spleen and rat liver [15], human liver [16] and ox
kidney [17]. We have artificially created a dimer of rat

BVR-A by using fused GST domains as sites for
dimerization [20]. The Synechocystis enzyme is there-
fore the first natural dimer reported for BVR-A. We
were careful to use a range of techniques to measure
the native molecular mass of sBVR-A and to conduct
these experiments under a range of conditions, includ-
ing temperature, pH and the presence or absence of
phosphate, because this has such a pronounced activat-
ing effect on the mammalian enzymes with NADH as
cofactor [14]. Under all of these conditions, the native
enzyme exhibits a molecular mass of 66–80 kDa and,
because the molecular mass is 34 kDa as measured by
SDS ⁄ PAGE, we conclude that sBVR-A is a stable
dimer. In light of the recent reports on the structures
of monkey dihydrodiol dehydrogenase [21] and the
N-terminally truncated dimeric form of glucose–fruc-
tose oxidoreductase [22], we propose that sBVR-A
joins this small family of pyridine nucleotide-depen-
dent oxidoreductases that dimerize via the C-terminal
b-sheet domain. It is intriguing that we purify sBVR-A
with bound pyridine nucleotide because this is also a
feature of the glucose–fructose oxidoreductase enzyme.
In addition to its unique quaternary structure,
sBVR-A also exhibits a sharp pH optimum, which we
reproducibly measured as pH 5. This behaviour is in
contrast to that displayed by the mammalian BVR-A
monomers, which show activity over a broad range of
pH values in the range 5–9 [14]. The cyanobacterial
BVR-A is not subject to the potent substrate inhibition
observed with the mammalian forms and this has

Fig. 7. A model for sBVR-A. sBVR-A model (green) superimposed
on the rat BVR-A crystal structure (grey). The amino acid residues
that mutated and their positions within the sBVR-A model are
shown. The numbers indicated represent the amino acid residues
of sBVR-A.
J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A
FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4421
allowed us to complete a full initial rate study on the
Synechocystis enzyme and to rigorously establish that
it obeys an ordered steady-state mechanism. The effect
of pH on the initial rate parameters has allowed us to
identify two ionizing groups in the free enzyme that
are required in the unprotonated (pK 5.1) and proton-
ated forms (pK 6.1), respectively, for binding NADPH.
The protonation state of the lower pK does not affect
the binding of NADH. This is consistent with an ioniz-
ing residue, pK 5.1, in the free enzyme which, in the
deprotonated state, may promote interaction with the
2¢-phosphate group of NADPH but plays no signifi-
cant role in binding NADH. In the case of human
BVR-A, we have suggested that the protonation state
of Glu75, may effect the interaction with Arg44, which
is a key residue involved in binding the 2¢-phosphate
of NADPH [14]. Further work is required to identify
possible analogous candidates in sBVR-A. There is
clearly a pK of 5.4 in the ternary complex that is
required to be protonated for efficient catalysis with
NADPH and which, with NADH as cofactor, exhibits
apK of 5.7. Our mutagenesis studies tentatively iden-
tify this residue as His84. We have recently discussed

the possibility that a His residue may be responsible
for supplying a proton to the pyrrole nitrogen atom of
biliverdin-IXb prior to hydride transfer in the case of
human biliverdin-IXb reductase (hBVR-B). Although
structurally distinct to BVR-A, BVR-B is a good
model for mechanistic studies on the reduction of the
linear tetrapyrrole ‘C10’ position by hydride. BVR-B is
a ‘non-Ixa’ biliverdin reductase [23] and is unable to
accommodate biliverdin-IXa in a productive orienta-
tion, although we have shown that it clearly binds,
albeit rotated by 90° [24], when compared with the bil-
iverdin isomers that are substrates (i.e. the IXb,IXd
and IXc isomers). Mutagenesis studies on BVR-B have
indicated that a solvent hydroxonium ion may be the
source of the proton and this was found to be consis-
tent with quantum mechanical ⁄ molecular mechanical
calculations [25]. However, our studies with BVR-B as
a model have demonstrated that there is a requirement
for proton transfer to the pyrrole nitrogen atom prior
to hydride transfer in the hBVR-B reaction co-ordinate
[25] and we suggest that His84 is a good candidate for
this function in sBVR-A. The second ‘more acidic’ pK
in the k
cat
data set (pK 4.7) is also prominent with
NADPH but less so with NADH.
We have taken advantage of the induced CD spectra
of biliverdin when enantiomeric forms are stabilized by
binding to proteins, including serum albumins [26]
and, as reported in the present study, sBVR-A. In

solution, these chiral forms are clearly in equilibrium
so that no CD spectrum is seen. Bilirubin adopts two
enantiomeric ‘ridge tile’ configurations [19,27], whereas
A
BC
D
E
Fig. 8. Induced CD spectra of biliverdin-IXa bound to sBVR-A and various mutants. sBVR-A and the mutants indicated (all at 29 lM) were
incubated with biliverdin-IXa (30 l
M) and NADP
+
(100 lM). (A) Wild-type, (B) H84A, (C) D285A, (D) E101A and (E) Y102A.
Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle
4422 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS
biliverdin is suggested to oscillate between two helical
‘lock washer’ configurations, and one of these, the P-
configuration, is clearly stabilized in a biliverdin–myo-
globin complex [28]. Although it is tempting to suggest
that trapping oscillations between two helical forms is
the phenomenon responsible for the P(lus) and
M(inus) spectra of biliverdin when bound to human
serum albumin (HAS) and BSA respectively [26], this
remains to be confirmed. A recent X-ray structure of
HSA with bilirubin bound [29] shows a ZZE configu-
ration (not a ridge tile), whereas, in solution, HSA
stabilizes a P-type induced CD spectrum so that, until
we have CD spectra of the appropriate crystals, abso-
lute assignments will not be possible. The wild-type
and most mutants that we have studied show P-behav-
iour [by convention the sign of the longer wavelength

defines (P)lus or (M)inus]. However the Y102A mutant
appears to stabilize the inverted chiral M-form. This
mutant exhibits catalytic activity (k
cat
is approximately
50% of wild-type), albeit with a seven-fold increase in
the K
m
BILIVERDIN
. Because the K
m
NADPH
is very simi-
lar to wild-type, this suggests that hydride transfer
from the C4 of the nicotinamide ring can be accom-
plished relatively efficiently, even with variable configu-
rations of biliverdin bound at the active site. The
Y102A mutant therefore accomodates a variant config-
uration of biliverdin to the wild-type enzyme but
retains the ability to catalyse the transfer of hydride
from both pyridine nucleotides. As discussed previ-
ously [30], the most likely model for the biliverdin
binding site can accommodate a number of conforma-
tions of biliverdin, including the various locked iso-
mers that have been shown to bind productively in
hBVR-A [30] and the two helical P- and M-conformers
described in the present study.
The description of a functional BVR-A in some
cyanobacteria introduces an important issue with
regard to the subcellular localization of both PcyA and

sBVR-A. These two enzymes potentially compete for
substrate and different subcellular localizations would
provide a way out of this hypothetical dilema. The opti-
mum pH for activity for sBVR-A at acid pH values is
consistent with the hypothesis that sBVR-A may be
localized in the lumen of the thylakoid [5], which is
reported to maintain a pH in the range 5.5–5 [31]. As a
result of the low abundance of this protein, we have not
been able to confirm this using immunogold labelling
(L. Weaver, J. M. Hayes & T. J. Mantle, unpublished
results). The enzymes responsible for the synthesis of
the light-harvesting pigments phycocyanobilin and phy-
coerythrobilin (PcyA, PebA and PebB) are all ferre-
doxin-dependent and their reaction product is destined
for incorporation into the phycobilisomes that decorate
the cytosolic side of the thylakoid membrane. The bilin
reductase PcyA exhibits a pH optimum of 7.5 [32],
whereas PebA and PebB are assayed at pH 7.5 [4], con-
sistent with a distinct subcellular localization to sBVR-
A and most likely the cytosol, which has been reported
to maintain a pH in the range 6.8–7.2 [31]. Further
work is required to resolve this important question.
Experimental procedures
The protein coding DNA for sBVR-A was amplified from
Synechocystis PCC6803 genomic DNA using forward (5¢-
CGCGGATCCCATGTCTGAAAATTTTG-3¢) and reverse
(5¢-CGCCTCGAGCTAATTTTCAACTATATC-3¢) primers
containing BamH1 and Xho1 sites, respectively, to allow
directional cloning into a modified pET41a expression vec-
tor (Novagen, Madison, WI, USA). The GST-sBVR-A

fusion protein expressed from pETBVR-A in E. coli BL21
(DE3) cells was purified on glutathione-sepharose (Chroma-
trin Ltd, Dublin, Ireland) cleaved with thrombin (Sigma–
Aldrich, St Louis, MO, USA) and the GST fragment
removed by affinity chromatography on glutathione-sepha-
rose. Prior to HPLC analysis, the purified protein was
incubated in 6 m urea at 95 °C for 2 min, centrifuged at
16 000 g for 2 min and immediately loaded onto a Supelco
Discovery C18 reversed phase HPLC column (Supelco,
Bellefonte, PA, USA) (25 · 4 mm) at a flow rate of
1mLÆmin
)1
. The HPLC column was equilibrated in
100 mm potassium phosphate (pH 6) and elution was
achieved using a linear gradient of 0–40% methanol.
Size-exclusion chromatography was conducted using
1 · 100 cm Sephacryl 200 HR (Sigma–Aldrich) columns
equilibrated at pH 5 (25 mm sodium citrate, 100 mm NaCl)
and pH 7.5 (25 mm Tris ⁄ HCl, 100 mm NaCl) at both 4 °C
and 20 °C. The calibration proteins used [b-amylase
(200 kDa), alcohol dehydrogenase (150 kDa), BSA
(66 kDa), carbonic anhydrase (29 kDa) and cytochrome c
(12.4 kDa)] were individually applied to the column and
their elution volumes used to construct a standard curve of
log molecular mass versus elution volume.
Light scattering was performed on sBVR-A at pH 5 and
pH 7.5 at 20 °C. Protein samples (0.25 mgÆmL
)1
) were clar-
ified using a 0.22 lm filter and applied to an S-200 Super-

dex HR gel-filtration column connected to an AKTA
FPLC system (Amersham Biosciences, Little Chalfont,
UK). The column was run at 20 °C and a flow rate of
0.5 mgÆmL
)1
in the desired equilibration buffer (25 mm
sodium citrate, pH 5, 100 m m NaCl or 25 mm Tris ⁄ HCl,
pH 7.5, 100 mm NaCl). The gel-filtration column was con-
nected online to a miniDawn Tristar light-scattering detec-
tor (Wyatt Technology, Santa Barbara, CA, USA) and an
Optilab rEX Rayleigh interference detector (Wyatt Tech-
nology). The weight-average molar mass of sBVR-A was
calculated using the software astra (Wyatt Technology).
J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A
FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4423
For sedimentation velocity experiments, sBVR-A samples
(0.42 mL of 0.1 mgÆmL
)1
) were centrifuged in an Optima
XL-I analytical ultracentrifuge (Beckman Instruments, Inc.,
Palo Alto, CA, USA) at 201 600 g and at a temperature of
11 °Cor20°C. Changes in solute concentration were
detected by Rayleigh interference and radial absorbance
scans at 287 nm. Results were analysed by whole-boundary
profile analysis using the software sedfit, version 9.4 (P.
Schuck, />htm). For sedimentation equilibrium experiments, sBVR-A
samples (0.1 mL) were centrifuged in a four-place An-60 Ti
analytical rotor running in an Optima XL-I analytical ultra-
centrifuge at a temperature of 4 °C at 11 612 g, 18 144 g
and 26 127 g sBVR-A concentrations used were 0.1, 0.3 and

1mgÆmL
)1
. Radial absorbance scans at 275 nm (ten repli-
cates, radial step size 0.001 cm) were performed at intervals
of 5 h. Interference scans were also collected. Scans were
considered to be at equilibrium by plotting radial offset ver-
sus time using the software winmatch (D. A. Yphantis,
The results were analy-
sed and the data were fitted to a one species model using a
least squares fitting routine or a monomer–dimer self-associ-
ating model using the software sedanal, version 4.34
(W. Stafford & P. Sherwood, />rasmb/ms_dos/sedanal-stafford/). The goodness of fit
parameter (rmsd) and fits with small, randomly distributed
residuals were used to assess the best fit to a particular
model. Buffer densities and viscosities were calculated using
the software sednterp [33].
Site-directed mutagenesis of pETBVR-A was carried out
as described previously [34]. sBVR-A and sBVR-A mutants
in 25 mm sodium phosphate (pH 7) containing 50 mm
sodium fluoride were analysed by CD using a Jasco-J815
CD spectrometer (Jasco Inc., Easton, MD, USA). To mea-
sure biliverdin binding to sBVR-A and sBVR-A mutants,
the induced CD spectrum of the linear tetrapyrrole was
recorded in 25 mm Tris ⁄ HCl (pH 7.5), 100 mm NaCl at a
protein concentration of 1 mgÆmL
)1
containing 10–30 lm
biliverdin-IXa (Chromatrin) and 100 lm NADP
+
(Calbio-

chem, San Diego, CA, USA).
BVR-A activity was measured by monitoring the increase
in absorbance at 460 nm as a result of the appearance of
bilirubin-IXa using a Hexios spectrophotometer (Thermo
Spectronic, Cambridge, UK) with online chart recorder
(Kipp & Zonen, Hilperton, UK). The typical reaction mix-
ture contained 1–5 lg purified sBVR-A, 100 mm sodium
citrate buffer (pH 5) and various concentrations of the sub-
strate biliverdin-IXa and cofactor NAD(P)H (Calbiochem).
The reaction was performed at 30 °C and initiated by the
addition of enzyme or NAD(P)H. The extinction coefficient
for bilirubin under these conditions is 35.75 mm
)1
Æcm
)1
.
For initial rate kinetics, data points (in triplicate) were fit-
ted to the Michaelis–Menten equation using a least squares
fitting routine and the computer software prism (GraphPad
Software Inc., San Diego, CA, USA) or wincurve fit,
version 1.3 (Kevin Raner Software, Victoria, Australia) to
obtain values for apparent V
max
and apparent K
m
. For
initial rate and product inhibition studies, data sets were
converted to double-reciprocal plots. To determine inhibi-
tion constants involving slope changes, the apparent
K

m
⁄ V
max
was replotted against the concentration of inhibi-
tory product. The straight line intercepted the x-axis at –K
is
to give the inhibition constant for the slope effect. For inhi-
bition constants involving intercept effects, the reciprocal of
the apparent V
max
was plotted against the concentration of
inhibitory product and the inhibition constant for the inter-
cept effect (K
ii
) was determined from the intersection on the
inhibitor concentration axis. For pH studies, determination
of pK values has been described previously [35,36].
Acknowledgements
We thank Tatsiana Rakovich and Kieran Crosbie-
Staunton for initial work on the pH kinetics. The
ultracentrifugation experiments were partly funded by
a grant from the Wellcome Trust to the Astbury
Centre for Structural and Molecular Biology at the
University of Leeds, UK. This work was funded by
Science Foundation Ireland and the Irish Research
Council for Science, Engineering and Technology.
References
1 Glazer AN (1985) Light harvesting by phycobilisomes.
Annu Rev Biophys Biophys Chem 14, 47–77.
2 Field CB, Behrenfeld MJ, Randerson JT & Falkowski P

(1998) Primary production of the biosphere: integrating
terrestrial and oceanic components. Science 281, 237–240.
3 Partensky F, Hess WR & Vaulot D (1999) Prochloro-
coccus, a marine photosynthetic prokaryote of global
significance. Microbiol Mol Biol Rev 63, 106–127.
4 Frankenberg N, Mukougawa K, Kohchi T & Lagarias
JC (2001) Functional genomic analysis of the HY2 family
of ferredoxin-dependent bilin reductases from oxygenic
photosynthetic organisms. Plant Cell 13, 965–978.
5 Schluchter WM & Glazer AN (1997) Characterization
of cyanobacterial biliverdin reductase. Conversion of
biliverdin to bilirubin is important for normal phyco-
biliprotein biosynthesis. J Biol Chem 272, 13562–13569.
6 Baranano DE, Rao M, Ferris CD & Snyder SH (2002)
Biliverdin reductase: a major physiologic cytoprotec-
tant. Proc Natl Acad Sci USA 99, 16093–16098.
7 Nakao A, Otterbein LE, Overhaus M, Sarady JK,
Tsung A, Kimizuka K, Nalesnik MA, Kaizu T,
Uchiyama T, Liu F et al. (2004) Biliverdin protects the
functional integrity of a transplanted syngeneic small
bowel. Gastroenterology 127, 595–606.
8 Nakao A, Neto JS, Kanno S, Stolz DB, Kimizuka K, Liu
F, Bach FH, Billiar TR, Choi AM, Otterbein LE et al.
Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle
4424 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS
(2005) Protection against ischemia ⁄ reperfusion injury in
cardiac and renal transplantation with carbon monoxide,
biliverdin and both. Am J Transplant 5, 282–291.
9 McDonagh AF (2001) Turning green to gold. Nat
Struct Biol 8, 198–200.

10 Kikuchi A, Park SY, Miyatake H, Sun D, Sato M,
Yoshida T & Shiro Y (2001) Crystal structure of rat
biliverdin reductase. Nat Struct Biol 8, 221–225.
11 Whitby FG, Phillips JD, Hill CP, McCoubrey W &
Maines MD (2002) Crystal structure of a biliverdin IX-
alpha reductase enzyme-cofactor complex. J Mol Biol
319, 1199–1210.
12 Kavanagh K, Elkins J, Ugochukwu E, Guo K, Pilka E,
Lukacik P, Smee C, Papagrigoriou E, Bunkoczi G,
Sundstrom M et al. Structural Genomics Consortium
(SGC). 2H63, PDB Release Date: 04-Jul-2006.
13 Elliott G & Mantle TJ (1995) Purification and proper-
ties of salmon liver biliverdin reductase. Biochem Soc
Trans 23, 389S.
14 Franklin E, Browne S, Hayes J, Boland C, Dunne A,
Elliot G & Mantle TJ (2007) Activation of biliverdin-
IXalpha reductase by inorganic phosphate and related
anions. Biochem J 405, 61–67.
15 Noguchi M, Yoshida T & Kikuchi G (1979) Purifica-
tion and properties of biliverdin reductases from pig
spleen and rat liver. J Biochem 86, 833–848.
16 Maines MD & Trakshel GM (1993) Purification and
characterization of human biliverdin reductase. Arch
Biochem Biophys 300, 320–326.
17 Phillips O & Mantle TJ (1981) Some kinetic and physi-
cal properties of biliverdin reductase. Biochem Soc
Trans 9, 275–278.
18 Florini JR & Vestling CS (1957) Graphical determina-
tion of the dissociation constants for two-substrate
enzyme systems. Biochim Biophys Acta 25, 575–578.

19 Trull FR, Ibars O & Lightner DA (1992) Conformation
inversion of bilirubin formed by reduction of the
biliverdin-human serum albumin complex: evidence
from circular dichroism. Arch Biochem Biophys 298,
710–714.
20 Ennis O, Maytum R & Mantle TJ (1997) Cloning and
overexpression of rat kidney biliverdin IX alpha reduc-
tase as a fusion protein with glutathione S-transferase:
stereochemistry of NADH oxidation and evidence that
the presence of the glutathione S-transferase domain
does not effect BVR-A activity. Biochem J 328, 33–36.
21 Carbone V, Sumii R, Ishikura S, Asada Y, Hara A &
El-Kabbani O (2008) Structure of monkey dimeric dihy-
drodiol dehydrogenase in complex with isoascorbic acid.
Acta Crystallogr D Biol Crystallogr 64, 532–542.
22 Lott JS, Halbig D, Baker HM, Hardman MJ, Sprenger
GA & Baker EN (2000) Crystal structure of a truncated
mutant of glucose-fructose oxidoreductase shows that
an N-terminal arm controls tetramer formation. J Mol
Biol 304, 575–584.
23 Cunningham O, Dunne A, Sabido P, Lightner D &
Mantle TJ (2000) Studies on the specificity of the tetra-
pyrrole substrate for human biliverdin-IXalpha reduc-
tase and biliverdin-IXbeta reductase. Structure-activity
relationships define models for both active sites. J Biol
Chem 275, 19009–19017.
24 Pereira PJ, Macedo-Ribeiro S, Pa
´
rraga A, Pe
´

rez-Luque
R, Cunningham O, Darcy K, Mantle TJ & Coll M
(2001) Structure of human biliverdin IXbeta reductase,
an early fetal bilirubin IXbeta producing enzyme. Nat
Struct Biol 8, 215–220.
25 Smith LJ, Browne S, Mulholland AJ & Mantle TJ
(2008) Computational and experimental studies on the
catalytic mechanism of biliverdin-IXbeta reductase. Bio-
chem J
411, 475–484.
26 Goncharova I & Urbanova
´
M (2008) Stereoselective bile
pigment binding to polypeptides and albumins: a circu-
lar dichroism study. Anal Bioanal Chem 392, 1355–1365.
27 Boiadjiev SE & Lightner DA (1997) Exciton chirality of
bilirubin homologs. Chirality 9, 604–615.
28 Wagner UG, Mu
¨
ller N, Schmitzberger W, Falk H &
Kratky C (1995) Structure determination of the biliver-
din apomyoglobin complex: crystal structure analysis of
two crystal forms at 1.4 and 1.5 A resolution. J Mol
Biol 247, 326–337.
29 Zunszain PA, Ghuman J, McDonagh AF & Curry S
(2008) Crystallographic analysis of human serum albu-
min complexed with 4Z,15E-bilirubin-IXalpha. J Mol
Biol 381, 394–406.
30 Franklin E, Browne S, Horan AM, Inomata K, Hammam
MAS, Kinoshita H, Lamparter T, Golfis G & Mantle TJ

(2009) The use of synthetic linear tetrapyrroles to probe
the verdin sites of human biliverdin-IXa reductase and
human biliverdin-IXb reductase. FEBS J, in press.
31 Belkin S, Mehlhorn RJ & Packer L (1987) Proton gra-
dients in intact cyanobacteria. Plant Physiol 84, 25–30.
32 Dammeyer T & Frankenberg-Dinkel N (2006) Insights
into phycoerythrobilin biosynthesis point toward meta-
bolic channeling. J Biol Chem 281, 27081–27089.
33 Laue T, Shah B, Ridgeway T & Pelletier S (1992) Com-
puter-aided interpretation of analytical sedimentation
data for proteins. In Analytical Ultracentrifugation in
Biochemistry and Polymer Science (Harding SE, Rowe
AJ & Horton JC, eds), pp 90–125. Royal Society of
Chemistry, Cambridge.
34 Fisher CL & Pei GK (1997) Modification of a
PCR-based site-directed mutagenesis method.
BioTechniques 23, 570–571.
35 O’Fagain C, Butler BM & Mantle TJ (1983) The effect
of pH on the kinetics of arylsulphatases A and B.
Biochem J 213, 603–607.
36 Phillips MF & Mantle TJ (1993) Inactivation of mouse
liver glutathione S-transferase YfYf (Pi class) by ethac-
rynic acid and 5,5¢-dithiobis-(2-nitrobenzoic acid).
Biochem J 294, 57–62.
J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A
FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4425