Biliverdin amides reveal roles for propionate side chains in bilin reductase recognition and in holophytochrome assembly and photoconversion

Article abstract of DOI:10.1021/bi100756x

Linear tetrapyrroles (bilins) perform important antioxidant and light-harvesting functions in cells from bacteria to humans. To explore the role of the propionate moieties in bilin metabolism, we report the semisynthesis of mono- and diamides of biliverdin IXα and those of its non-natural XIIIα isomer. Initially, these were examined as substrates of two types of NADPH-dependent biliverdin reductase, BVR and BvdR, and of the representative ferredoxin-dependent bilin reductase, phycocyanobilin:ferredoxin oxidoreductase (PcyA). Our studies indicate that the NADPH-dependent biliverdin reductases are less accommodating to amidation of the propionic acid side chains of biliverdin IXα than PcyA, which does not require free carboxylic acid side chains to yield its phytobilin product, phycocyanobilin. Bilin amides were also assembled with BV-type and phytobilin-type apophytochromes, demonstrating a role for the 8-propionate in the formation of the spectroscopically native P_r dark states of these biliprotein photosensors. Neither ionizable propionate side chain proved to be essential to primary photoisomerization for both classes of phytochromes, but an unsubstituted 12-propionate was required for full photointerconversion of phytobilin-type phytochrome Cph1. Taken together, these studies provide insight into the roles of the ionizable propionate side chains in substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences between the photoconversions of BV-type and phytobilin-type phytochromes.

Full text of DOI:10.1021/bi100756x

6070 Biochemistry 2010, 49, 6070–6082
DOI: 10.1021/bi100756x
Biliverdin Amides Reveal Roles for Propionate Side Chains in Bilin Reductase
Recognition and in Holophytochrome Assembly and Photoconversion†
Lixia Shang,‡ Nathan C. Rockwell,‡ Shelley S. Martin, and J. Clark Lagarias*
Department of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, California 95616.
‡These authors contributed equally to this work.
Received May 12, 2010; Revised Manuscript Received June 17, 2010
ABSTRACT: Linear tetrapyrroles (bilins) perform important antioxidant and light-harvesting functions in cells
from bacteria to humans. To explore the role of the propionate moieties in bilin metabolism, we report the
semisynthesis of mono- and diamides of biliverdin IXR and those of its non-natural XIIIR isomer. Initially,
these were examined as substrates of two types of NADPH-dependent biliverdin reductase, BVR and BvdR,
and of the representative ferredoxin-dependent bilin reductase, phycocyanobilin:ferredoxin oxidoreductase
(PcyA). Our studies indicate that the NADPH-dependent biliverdin reductases are less accommodating to
amidation of the propionic acid side chains of biliverdin IXR than PcyA, which does not require free carboxy-
lic acid side chains to yield its phytobilin product, phycocyanobilin. Bilin amides were also assembled with
BV-type and phytobilin-type apophytochromes, demonstrating a role for the 8-propionate in the formation of
the spectroscopically native Pr dark states of these biliprotein photosensors. Neither ionizable propionate
side chain proved to be essential to primary photoisomerization for both classes of phytochromes, but an
unsubstituted 12-propionate was required for full photointerconversion of phytobilin-type phytochrome
Cph1. Taken together, these studies provide insight into the roles of the ionizable propionate side chains in
substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences
between the photoconversions of BV-type and phytobilin-type phytochromes.
Heme-derived linear tetrapyrroles (bilins) perform important
roles as antioxidants and as light-harvesting and photosensory
pigments in many organisms (1, 2). Because of the ability of free
heme to generate strong intracellular oxidants in the presence
of oxygen, excess heme accumulation is averted by feedback
regulation of its biosynthesis and through its enzymatic conver-
sion to biliverdin IXR (BV)1 by heme oxygenases (3). Heme oxy-
genase is strongly product inhibited, so turnover is sustained by
coupled bilin reductase activity or by transfer to bilin-binding
proteins. The NAD(P)H-dependent biliverdin reductase A (BVR)
of animals is well tailored to this role, as formation of its lipo-
philic bilirubin IXR (BR) product is essentially irreversible (4, 5).
Oxygenic photosynthetic organisms can instead use ferredoxin-
dependent bilin reductases (FDBRs) to convert BV to the more
reduced phytobilins, phytochromobilin (PΦB), phycocyanobilin
(PCB), and phycoerythrobilin (PEB) (6, 7). The transfer of BV
from heme oxygenase to a bacteriophytochrome in Pseudomonas
aeruginosa (8) suggests that these widespread two-component
kinases contribute to heme turnover in eubacteria in addition to
their role as bilin and light sensors (9, 10).
An improved understanding of the substrate specificity of bilin
reductases is expected to presage the development of new reagents
of clinical and agricultural significance. In this regard, neonatal
jaundice arising from delayed induction of BR-specific UDP-
glucuronyl transferase expression is responsible for profound
neurological disorders in newborns. While treatment of neonatal
jaundice entails blue light exposure that triggers BR photo-
isomerization from the thermally favored 4Z,15Z form to produce
more readily excreted isomers such as 4Z,15E (11-13), treat-
ments for other hepatopathological and congenital forms of
bilirubinemia could benefit from new drugs that specifically
inhibit BVR activity (14). Inhibitors of FDBRs also are desirable
for use as potential algicides and as novel regulators of plant
growth and development. In cyanobacteria and some eukaryotic
algae, the phytobilin products of FDBRs are direct precursors
of the covalently bound chromophores of the abundant light-
harvesting phycobiliproteins (15, 16). The red (R) and far-red
(FR) light-sensing phytochromes use BV, PCB, or PΦB, whose
covalent association with a phytochrome apoprotein permits
†This work was supported by National Institutes of Health Grant
GM068552 to J.C.L., by National Science Foundation Center for
Biophotonics Science and Technology Subcontract PHY-0120999 to
J.C.L., and by a research award from the University of California Davis
NMR facility.
*To whom correspondence should be addressed. Telephone: (530)
752-1865. Fax: (530) 752-3085. E-mail: jclagarias@ucdavis.edu.
1Abbreviations: Δabsorbance, change in absorbance (in difference
spectra, which are reported as 15Z - 15E); BR, bilirubin IXR; BR13,
bilirubin XIIIR; BV, biliverdin IXR; BV-DA, biliverdin IXR diamide;
BV-8MA, biliverdin IXR 8-monoamide; BV-12MA, biliverdin IXR 12-
monoamide; BV13-DA, biliverdin XIIIR diamide; BV13-DA, biliverdin
XIIIR monoamide; CD, circular dichroism; DDQ, 2,3-dichloro-5,6-
dicyanobenzoquinone; DIEA, diisopropylethylamine; DME, dimethyl
ester; DMF, dimethylformamide; FDBR, ferredoxin-dependent bilin
reductase; FNR, ferredoxin-NADPþ reductase; HBTU, O-benzotri-
azole-N,N,N0,N0-tetramethyluronium hexafluorophosphate; HOBt, hydro-
xybenzotriazole; HPLC, high-performance liquid chromatography; MME,
monomethyl ester; PCB, phycocyanobilin; PCB-8MA, phycocyanobilin
8-monoamide; PCB-12MA, phycocyanobilin 12-monoamide; PΦB, phyto-
chromobilin; Pr, red-absorbing state of phytochrome; Pfr, far-red-absorbing
state of phytochrome, defined as a 15E chromophore red-shifted relative to
the 15Z state; TLC, thin layer chromatography. Holoproteins are described
throughout as the name of the protein followed by a colon and then by the
bilin, as in DrBphP:BV (the BV adduct of DrBphP).
r
pubs.acs.org/Biochemistry
Published on Web 06/21/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 29, 2010 6071
MATERIALS AND METHODS
their hosts to adapt to unfavorable light conditions (10, 17-20).
As potential antimicrobial agents and plant growth regulators,
bilin-based inhibitors of phytochrome biogenesis thus hold con-
siderable promise for both agriculture and medicine.
Reagents and General Procedures. All reagents and sol-
vents were ACS reagent-grade unless otherwise specified; HPLC-
grade solvents were used for reversed phase (RP-HPLC) chro-
matography. Bilirubin IXR (BR) was purchased from Frontier
Scientific (Figure 1A of the Supporting Information for rubin
structures). Bilirubin XIIIR (BR13) was synthesized according to
the method of Lightner and colleagues (46). Biliverdins were obta-
ined by oxidizing the corresponding bilirubins with 2,3-dichloro-
5,6-dicyanobenzoquinone (DDQ, Aldrich) as described by
McDonagh and co-workers (41, 47) using DMF as the solvent in
place of DMSO. Preparative column chromatography was
performed using Merck silica gel 60 (70-230 mesh). Analytical
thin layer chromatography (TLC) was performed on Merck 60
F254 plastic silica gel sheets (0.1 mm layer). Analytical RP-HPLC
(Figure 2 and Figure 2 of the Supporting Information) used a
Phenomenex Ultracarb ODS(20) column (250 mmꢀ4.6 mm, 5 μm
particle size) on an Agilent 1100 series HPLC system equipped
with an Agilent G1315B diode-array detector. A Phenomenex
Ultracarb ODS(20) column (250 mm ꢀ 10 mm, 5 μm particle size)
was used on the same HPLC system for preparative purification of
bilins. HPLC and TLC solvent systems used for the various com-
pounds under study are described in the sections pertaining to each
compound. Protein concentrations were determined with bicincho-
ninic acid (48) or Bradford (49) protocols using bovine serum albu-
min as a protein standard. Pigment concentrations were estimated
using previously reported extinction coefficients for the diacid.
Characterization of Bilin Amides. Compounds synthesized
in the course of this study were characterized by 1H NMR spec-
troscopy, UV-vis absorption spectroscopy, and mass spectro-
metry. Table 1 of the Supporting Information reports peak
absorbance wavelengths and m/z values. 1H NMR spectra were
recorded on a 500 MHz Bruker-500 spectrometer in d5-pyridine
(99.96%, Aldrich), and assignments are reported in Tables 2 and
3 of the Supporting Information. UV-visible spectra (Figure 3 of
the Supporting Information) were recorded in a methanol/5% (v/v)
concentrated HCl, 50% aqueous acetone/20 mM formic acid, or
40% aqueous acetone/20 mM formic acid mixture. Spectra in
acidic methanol were recorded on Hewlett-Packard 8453 or Cary
50 spectrophotometers, while spectra in aqueous acetone and
formic acid were obtained in real time during RP-HPLC analysis.
Mass spectra were recorded on an Applied Biosystems Electron/
Spray Q-trap mass spectrometer with methanol as a sample sol-
vent. For estimation of bilin pKa values, bilins (3.4 μM in 50 mM
tetramethylammonium chloride) were characterized by absor-
bance spectroscopy at various pHs (buffers at 200 mM). Potassium
phosphate (pH 3-7), tris(hydroxymethyl)methylamine (Tris-
HCl, pH 8-9.5), N-cyclohexyl-3-aminopropanesulfonic acid
(CAPS-KOH, pH 10-11.5), and methylamine-HCl (pH 12) were
used as buffers.
Phytochromes are attracting an increasing amount of atten-
tion as targets for protein engineering, due to their potential uses
as gene photoswitches, as fluorescent reporters, and in agricul-
tural biotechnology (21-27). In this regard, the cyanobacterial
phytochrome Cph1 was converted from a photochemically active
red/far-red photosensor into a photochemically inert, intensely
fluorescent protein by a single point mutation (23, 28). Similar
results have subsequently been obtained for several related pro-
teins using either directed evolution or site-directed mutagenesis
(26, 29-31). The photoswitching properties of such proteins arise
via a combination of the intrinsic photochemical properties of the
chromophore and the effects of the surrounding protein matrix,
so the use of site-directed mutagenesis to modify that protein
matrix has also provided invaluable information about the effect
of chromophore-protein interactions on the photocycle and on
subsequent transduction of the light signal (24, 30, 32-34). The
broad photosensing range of the phytochrome-related cyano-
bacteriochrome family reflects their molecular evolution and natu-
ral selection for new regulatory functions using a common bilin
precursor (35).
The converse experiment, in which the structure and function
of a holophytochrome are perturbed by modification of the chromo-
phore, has also been undertaken. Indeed, nature has already
performed this experiment, yielding phytochromes with BV-,
PΦB-, and PCB-derived chromophores. While more extensive
chromophore modifications require synthetic chemistry expertise
and hence are more labor-intensive than site-directed mutagenesis
(36-39), the ability to incorporate semisynthetic BV isomers
and unnatural phytobilins into plant phytochromes has proven
to be particularly insightful in probing bilin-protein interactions
(21, 40-44). Chromophore analogues can provide information
about the roles played by specific moieties that cannot be obta-
ined in other ways, and hence such studies provide a valuable
counterpoint to approaches based on mutagenesis. We previ-
ously used such an approach to explore the importance of the
bilin 12-propionate side chain in two model phytochromes, Cph1
from Synechocystis sp. PCC6803 and DrBphP from Deinococcus
radiodurans (45).
Over the years, many BV analogues have been synthesized, but
surprisingly, syntheses of simple BV amides have not been des-
cribed. Here, we report the semisynthesis of mono- and diamides
of BV and those of the unnatural XIIIR isomer, employing BR as
the starting material. These biliverdin amides were used for inves-
tigations that initially focus on their ability to serve as substra-
tes for the representative bilin reductases, BVR and the FDBR
phycocyanobilin:ferredoxin oxidoreductase (PcyA). Our studies
indicate that BVR is considerably more sensitive to amidation of
the propionic acid side chains than PcyA, which does not require
free carboxylic acid side chains for catalysis. We have also
exploited the ability of PcyA to reduce the 8- and 12-monoamides
of BV to their corresponding 3E/3Z-PCB phytobilin products for
comparative studies that aimed to examine distinct roles played
by the propionate side chains in phytochrome assembly and
photointerconversion between its red-absorbing (Pr) and far-red-
absorbing (Pfr) forms. Our results demonstrate the utility of such
compounds in studying bilin-protein interactions and provide
further support for the proposal that Cph1 and DrBphP have
different requirements for formation of Pfr.
Representative Procedures for Semisynthesis of Biliverdin
IXR Diamide (BV-DA), Biliverdin XIIIR Diamide (BV13-
DA), and Bilirubin Dimethyl Esters. Bilirubin IXR (BR, 50 mg,
0.09 mmol) was suspended in 10 mL of DMF under argon with
stirring. Di(isopropyl)ethylamine (DIEA, Aldrich; 120 μL, 8 equiv)
was added, and the solution was stirred for 10 min before addi-
tion of a mixture of solid O-benzotriazole-N,N,N0,N0-tetramethyl-
uronium hexafluorophosphate (HBTU, Novabiochem; 98 mg,
0.26 mmol, 3.0 equiv) and hydroxybenzotriazole (HOBt, Nova-
biochem; 40 mg, 0.26 mmol, 3.0 equiv) under argon. The reaction
mixture was stirred in the dark for 1 h or until the solution was

6072 Biochemistry, Vol. 49, No. 29, 2010
Shang et al.
F
IGURE 1: Synthesis of bilin 8-monoamides. BR13 and BR13 dimethyl ester were co-acid-scrambled to yield mixed rubins, including BR-
12MME. Amidation of free propionate side chains and subsequent oxidation yielded mixed verdins, including BV-8MA12MME. Ester cleavage
and purification yielded BV-8MA, which could be enzymatically converted to PCB-8MA by the ferredoxin-dependent bilin reductase PcyA.
clarified. Solid crystalline NH4Cl (Aldrich; 37 mg, 0.69 mmol,
8 equiv) was then added, and the solution was stirred in the dark
under argon for 10-12 h. The reaction mixture (containing a light
yellow precipitate) was then poured into a solution of 2,3-dichloro-
5,6-dicyanobenzoquinone (DDQ, 41 mg, 0.18 mmol, 2.1 equiv)
in DMF (70 mL) with stirring and argon bubbling. After 5 min,
argon-saturated water (400 mL) was added to stop the reaction,
and the green product was extracted several times with chloro-
form (100 mL) until the aqueous layer was colorless. The com-
bined chloroform layer was extracted with water (5 ꢀ 100 mL)
and once with brine (100 mL). The resulting organic phase was
evaporated to dryness to yield crude biliverdin IXR diamide (BV-
DA) as a dark green solid. Silica gel TLC [9:1 (v/v) CHCl3/MeOH]
showed one main spot (Rf=0.3). BV-DA was further purified by
gravity flow silica gel chromatography to afford 20 mg of final
product (40% yield). A similar procedure was used with BR13
as the starting material for synthesis of biliverdin XIIIR
diamide (BV13-DA). The bilirubin dimethyl esters, BR-DME
and BR13-DME, were also prepared by similarly activating the
corresponding bilirubin isomer with HBTU/HOBt followed by
introduction of methanol in excess.
In addition to proceeding with oxidation in a one-pot reaction,
we were able to isolate bilirubin diamide by adding excess water
to the reaction mixture and then collecting BR diamide as a
yellow solid via filtration. However, BR diamide exhibited extre-
mely poor solubility in a number of solvents, including chloro-
form, DMF, acidic DMSO, and water. The poor solubility of BR
diamide in DMSO is likely to explain its inert character in acid
coscrambling reactions.
Representative Procedure for Semisynthesis of Biliverdin
IXR 8-Monoamide (BV-8MA) and Biliverdin XIIIR Mono-
amide (BV13-MA). A mixture of BR-DME (40 mg, 0.07 mmol)
and BR XIIIR (BR13, 40 mg, 0.07 mmol) was dissolved in dimethyl
sulfoxide (DMSO, 30 mL). Concentrated hydrochloric acid
(3 mL) was rapidly added to the solution with stirring. After being
stirred for 5 min at room temperature under argon, the reaction

Article
Biochemistry, Vol. 49, No. 29, 2010 6073
mixture was poured into ice-cold water (300 mL) and stirred for
15 min. The resulting brown precipitate was collected by vacuum
filtration and washed with water (3ꢀ100 mL). The product was
dried over phosphorus pentoxide in a vacuum desiccator over-
night to afford mixed rubins (Figure 1) as crude product (70 mg,
∼0.12 mmol). Crude mixed rubins (70 mg) were suspended in
DMF (14 mL), and DIEA (100 μL, 5 equiv) was added with stir-
ring. After 10 min, HBTU (79 mg, 0.21 mmol) and HOBt (32 mg,
0.21 mmol) were directly added to the reaction mixture. The
solution was stirred for 1 h before solid ammonium hydrochlo-
ride (30 mg, 0.56 mmol) was added. After overnight incubation
with stirring in darkness under argon, the reaction solution
was poured into a solution of DDQ (58 mg, 0.25 mmol) in DMF
(100 mL). Argon-saturated water (500 mL) was added to the mix-
ture after 5 min, and products were isolated by repeated extrac-
tion with chloroform (100 mL each) until the aqueous layer was
colorless. The combined organic phases were washed with water
(3ꢀ100 mL) and evaporated to dryness to yield 40 mg of mixed
bilin products as a dark green solid. This mixture showed three
major blue-green spots on silica gel TLC (9:1 CHCl3/MeOH).
The fastest-eluting species (Rf = 0.45) corresponded to mixed
biliverdin dimethyl esters (group I); the middle species (Rf=0.25)
corresponded to monomethyl esters of both BV-8MA and BV13-
MA (group II), and the slowest-migrating species (Rf = 0.11)
contained biliverdin diamides (group III). The three components
were resolved by silica gel column chromatography (20:1 or 10:1
CHCl3/MeOH) to yield group I (4 mg), group II (10 mg), and
group III (4 mg). The group II biliverdin-MA monoester mixture
was subjected to the modified ester cleavage method of Lindner
et al. (50). This entailed dissolving the monoamide/monoester
mixture (4 mg, ∼7 μmol) in 50% aqueous trifluoroacetic acid
(TFA, 6.4 mL) to which prewashed DOWEX 50WX8-200 ion-
exchange resin (1.4 g) was added with stirring in darkness. Ester
cleavage was monitored by TLC (9:1 CHCl3/MeOH) and by
HPLC (mobile phase, 50% aqueous acetone containing 20 mM
formic acid). After hydrolysis had been completed (∼2 days), the
resin was removed by filtration and the filtrate was diluted with
distilled water (60 mL). The resulting solution was applied to a
C18 Sep-Pak solid phase cartridge (Waters) pre-equilibrated with
0.1% aqueous TFA. The loaded Sep-Pak column was washed
with water (10 mL) and then with 0.1% aqueous TFA (10 mL).
Biliverdin products were eluted with acetonitrile followed by
vacuum drying to yield biliverdin monoamides as a green solid.
Preparative HPLC was used to separate biliverdin XIIIR mono-
amide (BV13-MA, 0.3 mg, 0.5 μmol, 15% theoretical yield assum-
ing a 1:1 mix of monoamides) and biliverdin IXR 8-monoamide
(BV-8MA, 0.2 mg, 0.3 μmol, 10% theoretical yield).
F
IGURE 2: Reaction of BV with PcyA. BV derivatives were analyzed
by RP-HPLC in a 50% aqueous acetone/20 mM formic acid mixture
either with (red) or without (blue) prior treatment with PcyA.
Absorbance at 650 nm was monitored using a diode-array detector.
For the top spectrum, reduction of BV gave rise to a mix of 3E-PCB
and 3Z-PCB, as expected (52). For the spectrum second from the top,
reduction of BV-8MA gave rise to two product peaks (a and b)
identified as 3E-PCB-8MA and 3Z-PCB-8MA, respectively. For the
spectrum third from the top, reduction of BV-12MA produced
products c and d, similarly identified as 3E-PCB-12MA and 3Z-
PCB-12MA, respectively. Absorbance spectra for 3E- and 3Z-PCB
and products a-d are presented in Figure 6 of the Supporting
Information, and peak wavelengths are reported in Table 5 of the
Supporting Information. For the bottom spectrum, reduction of BV-
DA by PcyA resulted in formation of several apparent products that
were not well resolved in this mobile phase. Further characterization
of these products (Figure 7 of the Supporting Information) suggested
the presence of PCB diamide, but it was not possible to obtain pure
products.
DrBphP were each dialyzed against TKKG buffer [25 mM TES-
KOH (pH 8.5), 100 mM KCl, and 10% (v/v) glycerol]. For ex-
pression of mammalian (rat) BVR, E. coli strain BL21 was trans-
formed with pGEX6P1-ratBVR, a GE Healthcare GST gene
fusion plasmid, and grown in LB. Plasmid pGEX6P1-ratBVR
(generous gift of M. D. Maines) encodes GST-tagged BVR. This
plasmid was found to contain two mutations relative to the rat
BVR sequence established by X-ray crystallography (54): D263G,
which is a known polymorphism, and G141W. Neither mutation
is predicted to lie close to the active site. IPTG (1 mM) was used
to induce BVR expression. Clarified lysate was loaded onto a
glutathione affinity column equilibrated in phosphate-buffered
saline (PBS). After the mixture had been extensively washed with
PBS, BVR protein was eluted from the column in 50 mM Tris-
HCl (pH 8) supplemented with 10 mM reduced glutathione.
Eluted BVR was concentrated by centrifugal ultrafiltration (10K
MWCO) and dialyzed in phosphate buffer [100 mM potassium
phosphate (pH 7.4) and 10% (v/v) glycerol]. To obtain Synecho-
cystis biliverdin reductase [BvdR (59)], E. coli strain BL21 was
transformed with plasmid pASK75B-BvdR [obtained via inser-
tion of the PCR-amplified Synechocystis BvdR locus into the
Biometra Strep-tag vector pASK75B (55)] and grown in LB
medium. BvdR protein was expressed and purified using strep-
tag resin (IBA) in accordance with the manufacturer’s instruc-
tions. Purified protein was dialyzed against phosphate buffer
[100 mM potassium phosphate (pH 7.4) and 10% (v/v) glycerol].
After dialysis, all recombinant proteins were flash-frozen in liquid
nitrogen and stored at -80 °C prior to use.
Semisynthesis of Biliverdin IXR 12-Monoamide (BV-
12MA). A similar procedure was used as described above using
bilirubin XIIIR dimethyl ester (BR13-DME) and bilirubin IXR
(BR) as starting materials to generate biliverdin IXR 12-mono-
amide (BV-12MA).
Preparation of Recombinant Proteins. Recombinant PcyA
from Anabaena sp. PCC 7120 and recombinant ferredoxin from
Synechococcus sp. PCC7002 were expressed in Escherichia coli as
described previously (51, 52). PcyA was purified as a glutathione
S-transferase fusion protein on glutathione-agarose (Sigma) (51),
while ferredoxin was purified by ion-exchange chromatography
on DEAE-Sephadex (53). Plasmids pBAD-Cph1N514-CBD and
pBAD-DrBphPFL-CBD (45) were used for expression of the 514
N-terminal amino acids of Cph1 and of full-length DrBphP as
apoproteins, respectively. Purified PcyA, ferredoxin, Cph1, and

6074 Biochemistry, Vol. 49, No. 29, 2010
Shang et al.
Biliverdin Reductase Assays. Mammalian BVR assays were
performed in absorbance cuvettes containing 1 mL of 0.1 M Tris-
HCl buffer (pH 8.7), 0.1 mM NADPþ, 1 mM glucose 6-phosphate,
0.1 unit/mL glucose-6-phosphate dehydrogenase, 1 mg/mL BSA,
1.25-5 μM bilin substrate, and 34 nM BVR. BvdR assays were
performed in absorbance cuvettes containing 1 mL of BvdR
buffer [0.1 M citrate (pH 5.8), 10% (v/v) glycerol, 0.2 mg/mL
BSA, 100 μM NADPH, 1-5 μM bilin substrate, and 0.5-2 μM
BvdR]. Enzyme concentrations for BV IXR substrate controls
were adjusted to 17 nM. Reactions were initiated by addition of
enzyme, and absorbance was monitored at 450 nm at 20 s inter-
vals. All assays were performed at room temperature (25 °C) in
duplicate. The amount of rubin product formed was calculated
on the basis of an absorption coefficient at 450 nm of 53000 M-1
cm-1. Initial rates of reaction were determined at substrate con-
centrations of 1.25, 2.5, and 5 μM. The reaction velocity was linear
with substrate concentration in this regime, indicating that
the reaction was proceeding under kcat/KM conditions for all sub-
strates (i.e., that total enzyme concentration is approximately
equal to free enzyme concentration). The linear slope is therefore
approximately equal to kcat/KM times the total enzyme concen-
tration, permitting determination of kcat/KM under the assump-
tions that the enzyme is purified to homogeneity and is entirely
active. These assumptions do not affect the measurement of speci-
ficity, which is determined by the ratio of kcat/KM values.
Reaction of Biliverdin Amides with PcyA. PcyA was assayed
under steady-state conditions (52), with all solutions degassed
under vacuum prior to use in a closed vessel. Standard assays
used TKK buffer [25 mM TES-KOH (pH 8.5) and 100 mM KCl],
while Mes-NaOH (pH 6.0) was used for the reaction of BV
diamide with PcyA at pH 6. Assays contained 0.1 μM PcyA,
5 μM biliverdin, 0.025 unit/mL ferredoxin-NADPþ reductase
(FNR), 5 μM ferredoxin, 10 μM bovine serum albumin, and an
oxygen scavenging system (25 units/mL glucose oxidase, 50 mM
glucose, and 25 units/mL catalase). Reactions were initiated by
addition of NADPH from a 30 mM stock with immediate
incubation of the reaction tube at 30 °C. After 30 min, reaction
mixtures were iced. Crude bilins were extracted with a C18 Sep-
Pak C18 cartridge and eluted as described above, followed by
evaporation to dryness using a SpeedVac concentrator (Savant).
Analytical RP-HPLC analyses were performed as described
above with monitoring at 650, 480, and 380 nm. Complete UV-
visible spectra were obtained for peaks of interest. This procedure
was performed on a larger scale for preparative production of
PCB amides for assembly with apophytochromes.
(CD) spectra were recorded on a Chirascan CD spectrometer
(Applied Photophysics). For denaturation experiments, holo-
phytochrome (150 μL) was combined with concentrated guani-
dinium chloride (900 μL) and concentrated hydrochloric acid
(10 μL). The final guanidinium concentration always exceeded 6 M.
Denatured samples were typically characterized by absorbance
spectroscopy on the Cary 50 spectrophotometer with or without
illumination to examine photochemistry over a period of e10 min
to avoid unwanted side reactions associated with the low pH.
RESULTS
Semisynthesis and Characterization of Biliverdin Amides.
Bilirubin IXR (BR) is commercially available, is inexpensive, and
can readily be converted to BV via mild oxidation. We therefore
chose BR as the starting point for the synthesis of biliverdin
amides. BR could readily be converted to its diamide (BR-DA)
by activation of the carboxylate moieties with the coupling
reagent HBTU. It was possible to similarly generate the diamide
of BR13 (BR13-DA). Unfortunately, both rubin diamides pro-
ved to be difficult to characterize because of extremely low
solubility in several solvent systems. It was also not possible to
generate BR monoamides via co-acid scrambling of BR and BR-
DA (56), because BR-DA proved to be inert in this reaction, pre-
sumably because of its low solubility. We were able to generate
BV diamide (BV-DA) from BR via amidation and subsequent
oxidation in a one-pot reaction (representative procedure in
Materials and Methods), and BV-DA proved to be more soluble
and hence more amenable to study. A similar approach could be
used to prepare BV13-DA. Using smaller amounts of ammonium
chloride in this procedure resulted in formation of a mixture of
BV monoamides, but it was not possible to purify either mono-
amide satisfactorily after such reactions.
We therefore devised an alternative approach for preparing
BV monoamides in which BR and BR13 were used as starting
materials (Figure 1; detailed procedures are presented in Mate-
rials and Methods). BR was converted to its dimethyl ester (BR-
DME) and then co-acid-scrambled with BR13. This reaction
regenerated starting materials along with BR 12-monomethyl
ester (BR-12MME) and BR13 monomethyl ester (BR13-MME).
This mixture was then reacted with HBTU, HOBt, and ammo-
nium chloride as for synthesis of the diamide, with subsequent
oxidation in a one-pot reaction to yield a mix of biliverdin esters
and amides that included BV-8MA/12-MME and BV13-MA/
MME. After ester cleavage and purification by preparative
HPLC, this route yielded BV-8MA and BV13-MA. By changing
the starting materials in this scheme to BR and BR13 dimethyl
ester, we were similarly able to synthesize BV-12MA.
The purity of the resulting bilin amides was assessed by analytical
RP-HPLC (Figure 2 and Figure 2 of the Supporting Information).
All compounds were found to be of good quality. Compared with
the parent BV diacid molecules, the corresponding amides elute
with shorter retention times: BV diamides first, followed by
monoamides and then diacids. BV diamides were more soluble in
chloroform or in mixed aqueous/organic media (10% DMSO or
10% DMF) than their parent diacids. This is in marked contrast
to the equivalent chloroform-soluble rubins, as BR diamides
were largely insoluble in all solvents examined.
The structures of BV amides were confirmed by mass spectro-
metry, absorbance spectroscopy, and 1H NMR spectroscopy
(Figure 2 and Tables 1-3 of the Supporting Information). All
compounds had correct m/z values. Peak absorbance wave-
lengths and absorbance line shapes were essentially identical in
Assembly of Bilin Amides with Apophytochromes and
Characterization of Monoamide Adducts. In vitro assembly
reactions were performed in TKKG buffer as described pre-
viously (45), with tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) added to a final concentration of 1 mM immediately
before assembly reactions were set up. Apophytochromes (Cph1 or
DrBphP) were diluted in freshly prepared buffer to a final volume
of 1 mL, and bilin was added such that the apophytochrome
was present in 2-4-fold excess. After incubation in darkness for
2 h at room temperature, reaction mixtures were dialyzed in
TKKG buffer overnight at 4 °C. The presence of a covalently
attached chromophore following dialysis was determined by
SDS-PAGE and zinc blotting (Figure 4 of the Supporting Infor-
mation). Phytochrome photochemistry was assayed using a Cary
50 spectrophotometer equipped with a cuvette holder permitting
irradiation of the sample from above with light from a 75 W
xenon source used with appropriate filters (45). Circular dichroism

Article
Biochemistry, Vol. 49, No. 29, 2010 6075
BV diacids and amides (Figure 3 and Table 1 of the Supporting
Information), providing evidence that the bilin π system active in
absorbance spectroscopy is not intrinsically affected by the loss of
carboxylate moieties. Moreover, the 1H NMR spectra of mono-
amides and diamides showed almost no changes relative to the
parent diacids (Tables 2 and 3 of the Supporting Information).
The exceptions are the appearance of amide protons in the BV
amide spectra and the appearance of additional peaks in the 1H
NMR spectrum of BV13-MA relative to the spectra of BV13 or
BV13-DA (Table 3 of the Supporting Information). Such addi-
tional peaks are expected for the asymmetrical BV13-MA relative
to the symmetrical BV13 and BV13-DA isomers (Figure 2C of
the Supporting Information). The NMR and absorbance spectra
thus provide evidence that the aromatic π system is not substan-
tially affected by the amide substitutions.
Further support for this conclusion comes from estimation of
the pKa value of the bilin π system. We chose to evaluate this by
absorbance spectroscopy for the parent BV diacid and for BV
diamide. This approach can produce complicated results due to
conformational changes (57), so we took these measurements in
the presence of 50 mM tetramethylammonium chloride to mini-
mize the effects of the propionate or amide side chains on chromo-
phore geometry. We found that the peak wavelength for the
bilin blue (Soret) absorbance band exhibited a clear transition
consistent with an apparent single spectroscopically sensitive
titratable group for both compounds (Figure 5 of the Supporting
Information). We measured identical pKa values of 5.7 for bili-
verdin diacid and diamide. This pKa is close to that of pyridine
(5.2), consistent with titration of the proton responsible for the
positively charged bilin π system [formally the C-ring nitrogen
(Figure 1 of the Supporting Information)] and not with the
propionates, which are absent in the diamide. We therefore con-
clude that the substitution of one or two amides for propionates
has very little effect on the bilin π system per se and hence could
be expected to have little effect on its intrinsic behavior as a chromo-
phore, while permitting assessment of the importance of the
propionate side chains for bilin-protein interactions.
Biliverdin Amides as Substrates for NAD(P)H-Dependent
Biliverdin Reductases. In some organisms, biliverdins are meta-
bolized by biliverdin reductases that target the C10 position to
generate bilirubins. Mammalian bilirubin production mainly
relies on the activity of NAD(P)H-dependent biliverdin reductase
A (BVR) (58), an enzyme with an apparent ortholog designated
as BvdR in many cyanobacteria (59). While BVR and BvdR both
target biliverdin R isomers, they exhibit very different pH depen-
dence profiles (59-61). To assess the importance of the bilin pro-
pionates in substrate binding and catalysis by these NAD(P)H-
dependent reductases, we examined the reaction of biliverdin amides
with recombinant rat BVR and BvdR from the cyanobacterium
Synechocystis sp. PCC 6803 (59, 62). The limited water solubility
of biliverdin and its amides precluded satisfactory saturation
kinetics. However, we were able to determine Vmax/KM values,
which were used to calculate kcat/KM values by assuming that
total protein concentration is an accurate measurement of the
active enzyme concentration. The results demonstrate the impor-
tance of the 8- and 12-propionates for BVR and BvdR specificity
(Table 4 of the Supporting Information). Both enzymes exhibited
similar substrate preference for the various bilin amides. For
BVR, all three monoamide substrates exhibited slight defects of
5-10-fold relative to the physiological substrate, BV (Table 4 of
the Supporting Information). For BvdR, the three monoamides
exhibited slightly larger catalytic defects of 50-70-fold. Rubin
formation by either enzyme could not be detected for either BV
diamide or BV13 diamide within the sensitivity of our spectro-
photometric assay. We therefore conclude that one propionate is
sufficient to permit turnover by BvdR, but that loss of both pro-
pionates profoundly inhibits catalysis by both types of NAD(P)H-
dependent biliverdin reductase.
Biliverdin Amides as Substrates for the Ferredoxin-
Dependent Biliverdin Reductase PcyA. While BV is reduced
at the C10 position to yield BR in mammalian tissue, its reduction
by FDBRs in plants and cyanobacteria yields the phytobilins
PΦB, PCB, and PEB (6). To assess the importance of the BV
propionates in initial substrate binding (i.e., formation of the
Michaelis complex) and subsequent catalysis by this class of
enzymes, we examined the utilization of biliverdin amides by the
representative FDBR PcyA, for which there are available crystal
structures with and without bound bilin (63-66). PcyA readily
converted BV to PCB, which accumulated as a characteristic
mixture of the 3Z and 3E isomers (Figure 2). PcyA also proved to
be competent to convert monoamides BV-8MA and BV-12MA
to the corresponding PCB monoamides, again with a mix of the
3E and 3Z forms (Figure 2 and Figure 6 and Table 5 of the Sup-
porting Information). By contrast, PcyA was unable to utilize
BV-DA as a substrate under standard reaction conditions at pH
8.5 (data not shown). However, at a lower reaction pH, PcyA
appeared to sustain reduction of BV-DA to the corresponding
PCB-DA product mixture (Figure 2). Using a different HPLC
mobile phase (40% acetone/20 mM formic acid) to improve reso-
lution (Figure 7 of the Supporting Information), three main peaks
and at least four additional minor species could be resolved. The
majority species exhibited an absorbance spectrum similar to that
of the starting BV-DA (Figure 7 and Table 5 of the Supporting
Information), despite its apparent change in elution time. The
species whose spectra best matched that of PCB in this solvent
system were only present at low levels (<50% of total) and were
only poorly resolved from minor contaminants, so it was not
possible to obtain pure products for detailed characterization. On
the basis of these results, we conclude that PcyA can recognize
BV-DA as a substrate only at low pH but that its metabolism is
inefficient.
We also examined the ability of PcyA to utilize BV13-DA and
BV13-MA as substrates (Figure 2A of the Supporting Infor-
mation). While BV13 diacid was converted into the iso-PΦB
product with moderate yield, the diamide proved to be inert at
both low and high pH (Figure 2A of the Supporting Information
and data not shown). By contrast, BV13-MA gave rise to a
mixture of products upon reaction with PcyA. With biliverdins
containing a single endovinyl group, such as BV IXR, PcyA can
generate a mixture of 3E and 3Z products (Figure 2 and Figure
2B of the Supporting Information). BV13 contains two endovinyl
groups (Figure 2C of the Supporting Information), so PcyA
could convert either (or both) to an ethylidene. Since BV13 and
BV13-DA are both symmetrical (Figure 2C of the Supporting
Information), because of their equivalent endovinyl groups, only
two products (3E and 3Z) are expected upon two-electron
reduction. BV13-MA is no longer symmetrical, so equivalent
reduction of one of the two endovinyl groups can give rise to four
possible products. In practice, it was not possible to resolve these
products from each other for detailed characterization. We conclude
that the propionate side chains are not critical for BV reduction by
PcyA, in keeping with the paucity of conserved interactions with
these moieties in FDBR crystal structures (63-66), but that at least
one propionate is important for efficient binding and/or catalysis.

6076 Biochemistry, Vol. 49, No. 29, 2010
Shang et al.
Assembly of Bilin Monoamides with Apophytochromes.
The efficient reduction of BV-8MA and BV-12MA by PcyA
allowed us to prepare PCB-8MA and PCB-12MA in sufficient
quantity for incorporation into phytochromes. Along with the
corresponding 8- and 12-monoamide isomers of BV, these com-
pounds enabled comparative studies of the roles of the propio-
nates in assembly and photochemistry of both BV-type (DrBphP)
and PCB-type (Cph1) phytochromes. In a previous study, we
reported that bilin 12-monoamides could assemble with both
Cph1 and DrBphP apoproteins to yield novel “dual-Pr” ground
states, apparently arising from formation of two spectroscopi-
cally distinct species in equilibrium with each other (45). We also
reported that the dual-Pr states of the DrBphP-BV-12MA adduct
could be simultaneously converted to dual-Pfr states by irradia-
tion of either dual-Pr peak, indicating that equilibration of the
two states was rapid; photoconversion of the dual-Pfr states of
this adduct demonstrated that they were also in rapid equilibrium
with each other (45). To extend these results, both classes of apo-
phytochrome were incubated with the corresponding 8-mono-
amides (Figure 3), followed by overnight dialysis to remove any
residual chromophore that had not formed a covalent holophy-
tochrome adduct. BV-8MA and PCB-8MA were both able to
form covalent adducts with the appropriate apophytochrome
(Figure 4 of the Supporting Information), although formation of
the DrBphP-BV-8MA adduct was clearly less efficient than
formation of DrBphP-BV and DrBphP-BV-12MA adducts.
As controls, assembly of DrBphP and Cph1 with diacid bilins
was shown to give rise to the expected enhancement of the red
absorbance band (Figure 3), consistent with adoption of the ex-
tended, protonated Pr structure (28, 67-70). This enhancement
was much weaker for the DrBphP-BV-8MA adduct (Figure 3B).
Indeed, the ratio of the red absorbance band intensity to the blue
absorbance band intensity (R:B ratio) was 2.6 for in vitro assem-
bled DrBphP with BV free acid, but only 0.5 with BV-8MA. The
peak absorbance wavelengths of the 8-monoamide adduct were
also blue-shifted relative to those of the diacid adduct (Table 6 of
the Supporting Information). The blue-shifted peak wavelengths
and very low R:B ratio of the DrBphP-BV-8MA adduct are
consistent with a cyclic and/or deprotonated chromophore (28).
Incorporation of PCB-8MA into apoCph1 resulted in forma-
tion of a covalent adduct with an even more unusual spectrum
(Figure 3E). The Cph1-PCB-8MA adduct exhibited a much
shorter peak absorbance wavelength than the diacid adduct
(Table 6 of the Supporting Information), with a reduced R:B
ratio and a pronounced long-wavelength shoulder. This stands in
marked contrast to native holophytochromes, which possess
short-wavelength vibronic shoulders (71). The long-wavelength
shoulder thus raises the possibility that the Cph1-PCB-8MA
adduct possesses a mix of multiple chromophore species, which
appears to be the case for the 12-monoamide adducts (45).
To further characterize monoamide adducts of phytochromes,
we characterized them by CD spectroscopy. The bilin π system is
CD active, and it is known that phytobilin phytochromes such as
Cph1 exhibit inversion of CD upon photoconversion from Pr to
Pfr, while bacteriophytochromes such as DrBphP do not (45).
Both DrBphP and Cph1 exhibit negative CD for the red band
and positive CD for the blue band in the Pr state, in keeping with
the similar Pr chromophore geometries observed in their crystal
structures (68, 70). DrBphP-BV-8MA and DrBphP-BV-12MA
adducts also followed this pattern (Figure 3C), with clear assign-
ment of both dual-Pr peaks of the DrBphP-BV-12MA adduct
being negative.
F
IGURE 3: Assembly of apophytochromes with bilin 8-monoamides.
(A) Absorbance spectra for free BV (blue) and BV assembled with
DrBphP and then dialyzed to remove unincorporated chromophore
(DrBphP-BV, red) Starting amounts of BV were equal. (B) Absor-
bance spectra for assembly of BV-8MA with DrBphP. Colors and
sample handling are as described for panel A. (C) Adducts formed by
assembly of DrBphP with BV (blue), BV-8MA (red), and BV-12MA
(green) analyzed by CD spectroscopy. (D) Absorbance spectra for
assembly of Cph1 with PCB. Colors, relative concentrations, and
sample handling are as described for panel A. (E) Absorbance spectra
for assembly of Cph1 with PCB-8MA. Colors and sample handling
are as described for panel A. (F) Adducts formed by assembly of
Cph1 with PCB (blue), PCB-8MA (red), and PCB-12MA (green)
analyzed by CD spectroscopy.
The Cph1-PCB-12MA adduct exhibited a similar CD spec-
trum for the dual-Pr ground state with both peaks exhibiting
negative rotations (Figure 3F). Surprisingly, the Cph1-PCB-
8MA adduct possessed a different CD spectrum with its red absor-
bance maximum exhibiting a positive rotation, and the blue band
exhibiting a negative rotation. This CD spectrum is similar to those
of PCB and 15Z-PVB adducts of phycobiliproteins (72-74).
We have proposed that PCB and BV CD spectra report disposition
of the D-ring on one face of the bilin ring system or the other (29, 45),
although other interpretations have also been proposed (75).
Interestingly, the region of the CD spectrum corresponding to the
longest-wavelength absorbance shoulder gave very weak signals
(Figure 3F), indicating the presence of either two species cancel-
ing each other out or a chromophore geometry associated with
very weak CD. The distinct behavior of this region of the spec-
trum is consistent with a mix of at least two Cph1-PCB-8MA
populations, one of which exhibits reversed CD relative to that
of the diacid Pr state. These results demonstrate that a free
8-propionate is required for normal formation of the Pr state in
both DrBphP and Cph1. In DrBphP, substitution of the appro-
priate 8-monoamide results in less efficient incorporation and

Article
Biochemistry, Vol. 49, No. 29, 2010 6077
formation of a cyclic adduct, while in Cph1, a mix of species
arises, including at least one species with the opposite CD.
The Propionates Are Nonessential for the Formation of
Pfr by DrBphP. We have previously demonstrated that the
dual-Pr state of the DrBphP-BV12MA adduct can readily photo-
convert to an apparent dual-Pfr state, while the dual-Pr state of
Cph1 cannot (45). We were interested in further characterization
of this process, so we examined its dual-Pfr state by absorbance
and CD spectroscopy (Figure 4A-C). In wild-type DrBphP,
both Pr and Pfr exhibit negative CD for the red (Q) band and
positive CD for the blue (Soret) band (45), with the Pfr
state exhibiting weaker CD signals than the Pr state. We found
that the DrBphP-BV-12MA adduct exhibits a similar pattern
(Figure 4C): both dual-Pfr peaks in the red/far-red region are
associated with negative CD, and the CD signals of dual-Pfr are
weaker than those of dual-Pr. The peak wavelengths measured in
absorbance and CD spectroscopy are in reasonable agreement
with each other (Table 6 of the Supporting Information). In this
study, most peak CD wavelengths for bands in the red/far-red
region were red-shifted slightly relative to the corresponding
absorbance peak wavelengths. This difference was also observed
when CD spectra were compared to the absorbance spectra
acquired simultaneously on the CD spectrometer itself (data not
shown), indicating that it does not arise due to differences in
machine calibration.
To assess the importance of the 8-propionate in DrBphP
photoconversion, we examined the response of the thermally
stable DrBphP-BV-8MA state to illumination of the red absor-
bance peak (Figure 4D-F). Interestingly, this illumination gave
rise to enhanced red absorbance on either side of the illumination
region, including far-red absorbance. This enhancement in red
absorbance was balanced by a loss of absorbance in the Soret
region, consistent with the idea that illumination produced either
a more extended conformation or an increase in the ratio of ex-
tended to cyclic species in a mixed population. The photochemi-
cal difference spectrum shows the appearance of product(s) at
650 and 754 nm, with the latter being identical to the Dr-BV Pfr
wavelength (Table 6 of the Supporting Information). CD spec-
troscopy showed that this long-wavelength peak was associated
with very weak but detectable negative CD, consistent with
formation of a small amount of an authentic Pfr species. Inter-
estingly, the region of the CD spectrum associated with the photo-
chemical product at 650 nm exhibited a different peak wavelength
(634 nm) and markedly different line shape relative to those of the
absorbance spectrum, raising the possibility that the spectra reflect
multiple components in this region.
F
IGURE 4: Photochemistry of DrBphP assembled with bilin mono-
amides. DrBphP-BV-12MA (A-C) and DrBphP-BV8-MA(D-F)
adducts were characterized by absorbance and CD spectroscopy.
(A) The DrBphP-BV-12MA adduct was converted from dual-Pr (blue)
to dual-Pfr (red) with 600 nm light ((5 nm), giving the difference
spectrum shown in panel B. (C) Both dual-Pr (blue) and dual-Pfr
(red) were characterized by CD spectroscopy. Inversion of CD upon
photoconversion was not observed, with both dual-Pfr red-band
peaks exhibiting negative CD. (D) The DrBphP-BV-8MA adduct
(blue) was illuminated with 670 nm light ((20 nm), giving enhanced
red absorbance and reduced blue absorbance (red). (E) The difference
spectrum is shown for the spectra presented in panel D. (F) Both the
DrBphP-BV-8MA dark state (blue) and photoproduct (red) were
characterized by CD spectroscopy. The photoproduct region cor-
responding to the Pfr state of the DrBphP-BV adduct was asso-
ciated with negative CD, indicating no inversion of CD occurred.
The photoproduct CD peak at shorter wavelength does not match
the line shape of the corresponding absorbance peak well (compare
to panel D), and the two peak wavelengths are not in very good
agreement (Table 6 of the Supporting Information). Assignment of
this peak is therefore uncertain, as discussed in the text.
In both DrBphP-BV-8MA and DrBphP-BV-12MA adducts,
the amount of product formed at actual Pfr wavelengths was
apparently smaller than that formed at other wavelengths. Such
substoichiometric formation of far-red absorbance has also been
reported in PcyA, in which it is not associated with photoiso-
merase activity but instead reflects the formation of lactim tauto-
mers of the bilin chromophore (52, 76). We therefore sought to
demonstrate the existence of an authentic 15E chromophore
in the Pfr states of the monoamide adducts. Denaturation of the
holoprotein adduct under acidic conditions provides a useful assay
for this purpose, both because of changes in peak wavelengths upon
denaturation and because the 15E photoproduct can be photo-
converted to the thermally stable 15Z form under acidic condi-
tions (73, 74). We therefore examined the response of DrBphP-
BV, DrBphP-BV-8MA, and DrBphP-BV-12MA adducts to
acidic guanidinium chloride (Figure 5).
Denaturation of the three DrBphP adducts in the thermally
stable state gave rise to very similar spectra (Figure 5), indicating
that the distinct Pr spectra of DrBphP-BV-8MA and DrBphP-
BV-12MA adducts reflect structural differences that do not per-
sist upon denaturation. Denaturation of the Pfr states of DrBphP-
BV and DrBphP-BV-12MA adducts also produced nearly
identical blue-shifted spectra, consistent with the formation of
the 15E isomer (73, 74). These results also demonstrate that the
dual-Pfr species of the DrBphP-BV-12MA adduct collapsed into
a single species upon denaturation, as was the case for the two
Pr species of this adduct. Photoconversion of the denatured Pfr
DrBphP-BV and DrBphP-BV-12MA adducts resulted in con-
version of these species to species that were equivalent to those
obtained by denaturation of the Pr states. The difference spectra
for photoconversion of these adducts were strikingly similar

6078 Biochemistry, Vol. 49, No. 29, 2010
Shang et al.
photochemical difference spectrum (Figure 6B). Interestingly,
irradiation of this adduct at shorter wavelengths produced a
much smaller amount of the 705 nm product (despite greater light
absorption at this wavelength) along with little to no product at 628
nm (Figure 6B and data not shown). This differential product
formation as a function of illumination wavelength is consistent
with our hypothesis that the Cph1-PCB-8MA adduct contains
multiple populations of chromophore, although we cannot rule
out the possibility that the shorter-wavelength irradiation yields a
reduced Pfr:Pr photoequilibrium ratio due to greater overlap of Pr
and Pfr states.
A similar analysis of the Cph1-PCB-12MA adduct was under-
taken to see whether this adduct might also contain such a mix-
ture. As previously reported, illumination of the Cph1-PCB-12MA
adduct with 650 ( 20 nm light resulted in depletion of both dual-Pr
peaks, with no loss of absorbance in a narrow region at approxi-
mately 610 nm (Figure 6C). Irradiation of this adduct with 550 (
35 nm light produced a smaller difference spectrum (Figure 6D),
but with similar features: depletion of both dual-Pr absorbance
peaks and a similar constant absorbance at 610 nm. Thus, by con-
trast to the different populations of the Cph1-PCB-8MA adduct,
the two populations of the Cph1-PCB-12MA adduct produce
similar photochemical products.
The unchanging absorbance at 610 nm upon illumination of
the Cph1-PCB-12MA adduct raised the possibility that this
adduct produced a bona fide 15E bilin with peak absorbance in
this region. To test this possibility and to further characterize the
Cph1-PCB-8MA adduct, we subjected both adducts and wild-
type Cph1-PCB to acidic denaturation (Figure 7 and Table 7 of
the Supporting Information). The Pr and Pfr states of all three
adducts gave rise to comparable absorbance spectra, and photo-
conversion of all three denatured Pfr samples resulted in forma-
tion of products with spectra equivalent to those of the denatured
Pr samples. The resulting photochemical difference spectra were
equivalent (Figure 7D), indicating that all three chromophores
were able to undergo primary photoisomerization in native Cph1
to give rise to 15E bilins. However, the Cph1-PCB-12MA adduct
is unable to form detectable Pfr after primary photochemistry
occurs, indicating a requirement for the 12-propionate side chain
in subsequent thermal steps leading to formation of stable Pfr. The
Cph1-PCB-8MA adduct may be able to form very small amounts
of Pfr, although the difficulty in producing PCB monoamides in
large amounts precludes further characterization at this time.
F
IGURE 5: Denaturation analysis of DrBphP adducts. (A) The
DrBphP-BV adduct was denatured in acidic guanidinium chloride in
the native Pr (blue) or Pfr (green) state. Illumination of the denatured
Pfr state with 600 ( 5 nm light resulted in formation of a product (red)
whose absorbance spectrum was equivalent to that of the denatured Pr
sample. Similar results could be obtained by illumination of a dena-
tured Pfr sample with 650 (20 nm light (not shown). (B) Similar results
were obtained with the DrBphP-BV-12MA adduct. Here, illumina-
tion with 650 ( 20 nm light is shown; illumination with 600 ( 5 nm
light (not shown) gave similar results. (C) Similar experiments were
performed with the DrBphP-BV-8MA adduct. The spectrum of the
denatured Pfr DrBphP-BV-8MA adduct seemed to be a mix of the de-
natured Pr and Pfr spectra seen with other adducts, suggesting that
primary photochemistry was less efficient with the DrBphP-BV-8MA
adduct than with other adducts. (D) Difference spectra are shown for
photoconversion of the denatured Pfr adducts of DrBphP-BV (blue),
DrBphP-BV-12MA (green), and DrBphP-BV-8MA (red) adducts.
(Figure 5D), with very close peak/trough wavelengths (Table 7
of the Supporting Information). Denaturation of the DrBphP-
BV-8MA adduct in the Pfr state resulted in a somewhat different
spectrum (Figure 5C), although some absorbance did appear at
shorter wavelengths. However, illumination of this sample resul-
ted in formation of a spectrum identical to that of the denatured
Pr DrBphP-BV-8MA adduct, with a difference spectrum com-
parable to those of the DrBphP-BV and DrBphP-BV-12MA
adducts (Figure 5D and Table 7 of the Supporting Information).
We therefore conclude that the DrBphP-BV-8MA adduct is
capable of forming the 15E photoproduct, albeit less efficiently.
The equivalent photochemical difference spectra observed for
denatured DrBphP-BV, DrBphP-BV-8MA, and DrBphP-
BV-12MA adducts are consistent with a single primary photo-
chemical event in all three adducts. Taken together with the spec-
troscopic characterization of the native adducts, we conclude that
both DrBphP-BV-8MA and DrBphP-BV-12MA adducts are
able to form some Pfr, implying that neither propionate side chain
is essential for Pfr formation by DrBphP.
The Propionates Are Dispensable for Photoisomerization
in Cph1. We have previously shown that the Cph1-PCB-12MA
adduct is incapable of forming stable, detectable Pfr (45). To
examine photoconversion in the Cph1-PCB-8MA adduct, we
illuminated it with several light sources matching regions corres-
ponding to different populations detected by CD spectroscopy
(see above). Irradiation of the long-wavelength shoulder of this
adduct with 650 ( 20 nm light resulted in detectable photo-
chemistry (Figure 6A), with a main product band appearing
at 628 nm and a smaller band appearing at 705 nm in the
DISCUSSION
Using co-acid scrambling of appropriate rubin precursors with
subsequent oxidation, we have successfully synthesized BV
monoamides to examine the roles of tetrapyrrole propionic acid
side chains in ligand-protein interactions. BV monoamides are
well-suited to such experiments, because they do not exhibit
radically different solubility than the parent bilins in aqueous
buffers and because amidation of one or both side chains seems
to have minimal effects on the electronic structure of the tetra-
pyrrole π system as judged by NMR spectroscopy, absorbance
spectroscopy, and pKa measurements. Because the FDBR PcyA
can readily convert BV monoamides into PCB monoamides,
comparative studies using bilin monoamides in phytochromes
thought to differ in their Pr-Pfr interconversion are also enab-
led (45). Interestingly, we were unable to generate monoamides
via co-acid scrambling of BR diamide, because it exhibited very
low solubility, which may explain why it also proved to be inert in
co-acid scrambling reactions. Bilirubin itself is known to form

Article
Biochemistry, Vol. 49, No. 29, 2010 6079
F
IGURE 7: Denaturation analysis of Cph1 adducts. (A) The Cph1-
F
IGURE 6: Photochemistry of Cph1 assembled with PCB monoamides.
PCB adduct was denatured in acidic guanidinium chloride in the
native Pr (blue) or Pfr (green) state. Illumination of the denatured Pfr
state with 600 ( 5 nm light resulted in formation of a product (red)
whose absorbance spectrum was equivalent to that of the denatured
Pr sample. (B) Similar results were obtained with the Cph1-PCB-
12MA adduct. The apparent absorbance peak at approximately
800 nm in panels A and B is associated with a contaminant in some
lots of guanidine hydrochloride and is photochemically inert (not
shown). (C) Similar results were obtained with the Cph1-PCB-8MA
adduct. (D) Difference spectra are shown for photoconversion of the
denatured Pfr Cph1-PCB (blue), Cph1-PCB-12MA (green), and
Cph1-PCB-8MA (red) adducts.
(A) The Cph1-PCB-8MA adduct (blue) was illuminated with 650 (
20 nm light to form photoproducts (red). (B) The difference spec-
trum for 650 ( 20 nm illumination (blue) revealed the appearance of
products at 628 and 705 nm (Table 6 of the Supporting Infor-
mation). The difference spectra obtained upon illumination with
600 ( 5 nm light (green) displayed different product ratios. (C) The
Cph1-PCB-12MA adduct (blue) was illuminated with 650 ( 20 nm
light to form photoproducts (red). (D) The difference spectra
obtained upon illumination of the Cph1-PCB-12MA adduct with
650 ( 20 (blue) or 550 ( 35 nm light (green) indicated the presence
of comparable changes.
intramolecular hydrogen bonds between the propionate side
chains and the lactam moieties of the A- and D-rings in solution,
giving rise to the ridge-tile conformation in organic solvents (77).
BR diamide can still form such hydrogen bonds; indeed, the side
chain amides and the lactams can both supply donors and accep-
tors in BR diamide. Were such hydrogen bonds to form inter-
molecularly in BR diamide, the result could be poor solubility, as
we have observed.
deprotonated propionate). The requirement for at least one free
propionate is consistent with the extensive literature on the sub-
strate specificity of mammalian BVRs (78-84).
In contrast with the NAD(P)H-dependent enzymes, the FDBR
PcyA is less specific for substrates with free propionate side
chains. BV monoamides are quite good substrates, giving effec-
tively quantitative production of PCB monoamides. Moreover,
PcyA can also catalyze reactions with the diamide analogue of
BV; however, reduction of BV-DA yields a mixture of unnatural
products, and the reaction requires a lower pH. PcyA was unable
to metabolize the unnatural BV13-DA isomer, which likely ref-
lects its reduced binding affinity compared with that of the natu-
ral IXR isomer. The complex product mixture observed with
BV13-MA as a substrate appears to reflect distinct modes of
binding to the enzyme. PcyA binds its substrate in a cyclic C5-Z,
syn C10-Z,syn C15-Z,syn configuration, with the propionates
facing out of the active site into solvent (64). In this configuration,
BV13 could bind in either of two orientations, with the 3-vinyl
group or the 17-vinyl group occupying the position of the 3-vinyl
group of BV. For the symmetrical diacid, the two orientations
would give rise to only two products, i.e., 3Z and 3E isomers of
isoPΦB. By contrast, BV13-MA is asymmetric (Figure 2C of
the Supporting Information), so the two PcyA-binding orienta-
tions are expected to produce at least four distinct products if
only one vinyl group is reduced. It is thus not surprising that
BV13-MA gave rise to a number of products upon being treated
with PcyA.
NADPH-Dependent Biliverdin Reductases Are Less
Accommodating to Amidation of the Propionic Acid Side
Chains of Their BV Substrate Than the FDBR PcyA. We
have assessed BV monoamides as substrates for representative
NAD(P)H-dependent and ferredoxin-dependent biliverdin reduc-
tases. With rat BVR, substitution of either propionate resulted in
a modest catalytic defect of approximately 10-fold, while biliverdin
diamides were not metabolized. Cyanobacterial BvdR was also
unable to recognize diamides as substrates; additionally, this enzyme
exhibited more substantial catalytic defects (>50-fold) for reduc-
tion of the monoamides. The lack of reaction with either enzyme
with biliverdin diamides indicates that one of the propionic acid
side chains must be ionizable for efficient binding to, or catalysis
by, this family of enzymes. The lower pH optimum of BvdR (59)
implies possible protonation of titratable groups in the substrate
and/or active site, perhaps explaining the lower activity of this
enzyme compared with BVR (Table 4 of the Supporting Infor-
mation). The more severe defect observed in the reduction of mono-
amide substrates by BvdR than by BVR could arise for a similar
reason: protonation of a single propionate in the monoamide at
the lower pH optimum effectively mimics the inactive diamide
substrate. Thus, with BVR, the high pH means that the propio-
nate in the monoamide is more charged on average than at the
low pH of BvdR. The lower activity of BvdR with monoamides
thus reflects a lower concentration of the usable substrate (with
Phytochrome Assembly Experiments Reveal Distinct Roles
for 8- and 12-Propionates in Assembly and Photoconver-
sion of DrBphP and Cph1. The efficient production of PCB
monoamides by PcyA has facilitated comparative examination of
the roles played by the 8- and 12-propionates in two representa-
tive phytochromes, Cph1 and DrBphP. Both exhibit character-

6080 Biochemistry, Vol. 49, No. 29, 2010
Shang et al.
istic red/far-red reversible Pr-Pfr interconversions when associ-
ated with their natural chromophores, PCB for Cph1 and BV for
DrBphP. Like plant phytochrome, Cph1 exhibits inversion of the
red band CD upon formation of the Pfr state, while DrBphP and
other BphPs do not (45, 67, 75, 85). Additionally, we have
previously shown that the 12-propionate is required for forma-
tion of stable Pfr in Cph1, but not DrBphP. Thus, these two
phytochromes serve as representatives for the two known classes
of red/far-red photochemistry utilizing the knotted PAS-GAF-
PHY photosensory core module (45).
The 12-propionate specifically tunes the spectral region sensed in
the dark state and also is required for formation of a bona fide Pfr.
This implicates the 12-propionate in one or more of the light-
independent steps that occur after initial formation of the 15E
photoproduct to form Pfr in Cph1, which contrasts with no
critical involvement for the 12-propionate in DrBphP.
The 8-propionate appears to be critical to the spectral range
sensed by Cph1 in the Pr state. Additionally, the 8-monoamide Pr
adduct possesses at least one population with inverted CD rela-
tive to the diacid and 12-monoamide adducts. Such an inversion
suggests that the 8-monoamide D-ring is located on the opposite
face of the bilin ring system, filling space normally occupied only
in the Cph1 Pfr state (45, 68). Alternately, this signal could arise
due to a drastic twist about C5, moving the A-ring much farther
to the R-face such that the positive CD signal from the R-facial
A-ring is able to override the negative signal from the R-facial
D-ring, or due to a “flipped” population that places the D-ring in
the pocket normally occupied by the A-ring. This would invert
the facial disposition of the D-ring and hence the CD of the
chromophore. While such a population is not expected to be co-
valently bound, it remains formally possible that such a con-
formation could bind with sufficient affinity that it would remain
associated with the Cph1 protein during dialysis.
Future Perspectives. Our results indicate that bilin mono-
amides are useful research tools for characterizing protein-
ligand interactions in biliproteins. They also show that the roles
played by the propionates can vary within phytochromes, empha-
sizing the fact that red/far-red sensing by this important family of
photosensory proteins can take place in more than one way.
Fluorescent phytochrome mutants also hold promise as tools for
in vivo imaging (21, 24, 26). Engineering such phytochromes to
incorporate bilin diamides efficiently and preferentially might
prove to be a valuable addition to such approaches, because the
diamide chromophores are resistant to turnover by mammalian
biliverdin reductases and hence may offer improved stability and
sensitivity in imaging of mammalian tissue.
In DrBphP, both monoamides are able to support formation
of a small amount of the bona fide Pfr state as judged by absor-
bance and CD spectroscopy and by denaturation analysis.
However, neither exhibits normal photochemistry. In this regard,
the DrBphP-BV-12MA adduct exhibits dual-Pr and dual-Pfr, so
the actual ability to sense the ratio of red to far-red light is altered.
By comparison, the DrBphP-BV-8MA adduct adopts a more
cyclic Pr state and also generates multiple products in the Pfr state.
Denaturation analysis demonstrates that the primary photo-
chemistry is the same as that of the parent diacid adduct for both
monoamide adducts, but this photoisomerization seems less effi-
cient for the 8-monoamide adduct, possibly due to the altered
ground-state conformation. This analysis also demonstrates that
the dual peaks seen in the 12MA adduct collapse to a single peak
upon denaturation, indicating that they differ in a property that is
sensitive to denaturation, such as the degree of protonation or a
thermally accessible isomerization, but are not due to some pro-
perty that is unchanged by denaturation in aqueous acid, such as
a shorter conjugated system, e.g., phycoviolobilin or phycoery-
throbilin. In phytochromes utilizing BV as a precursor, such as
DrBphP, crystal structures provide good evidence that pri-
mary photochemistry occurs at the 15/16 bond (70, 86, 87).
Therefore, neither propionate is required for photoisomerization of
the 15/16 double bond in DrBphP, but both play roles in tuning the
interconversion between Pr and Pfr. The 8-propionate is thus
important for formation of the extended, red-enhanced Pr state
and hence is very important for sensing red light physiologically,
while the 12-propionate narrows the region that is sensed and
reduces spectral overlap between the Pr and Pfr states.
For Cph1, we previously showed that the 12-monoamide
adduct does not sustain formation of stable Pfr (45), even though
its primary photochemistry proceeds efficiently (Figures 6 and 7).
NMR and vibrational spectroscopy provide good evidence that
the primary photochemistry entails Z-to-E isomerization of the
15/16 double bond (39, 88-91), although a controversial recent
report has argued for photoisomerization of the 4/5 double
bond in a “knotless phytochrome” of the Cph2 type (19, 92, 93).
The 8-monoamide adduct gives rise to a complicated mixture
in the ground state (Figure 3). The components of this mixture
apparently have different photochemical responses (Figure 7),
with at least one of these components giving rise to a very small
amount of a species with a peak wavelength comparable to that
of Pfr for the diacid chromophore. We have not been able to char-
acterize the Cph1 monoamide photoproducts by CD spectros-
copy due to the spectral overlap of the Pr and Pfr states. However,
denaturation analysis confirms that both monoamide adducts
are able to undergo apparently normal primary photochemistry,
thus indicating that the multiple species observed in the ground
state collapse to a single, common conformation upon denatura-
tion. Therefore, like DrBphP, neither propionate is required for
double bond photoisomerization for Cph1. Nevertheless, both
propionates influence the interconversion between Pr and Pfr.
ACKNOWLEDGMENT
We thank Prof. Richard Vierstra (University of Wisconsin,
Madison, WI) for the original DrBphP expression construct,
Abigail Jang for construction of plasmid pBAD-Cph1FL-CBD,
and Alex King for technical assistance during this project.
SUPPORTING INFORMATION AVAILABLE
Seven figures and seven tables. This material is available free of
charge via the Internet at http://pubs.acs.org.
REFERENCES
1. Frankenberg, N. F., and Lagarias, J. C. (2003) Biosynthesis and
biological function of bilins. In The Porphyrin Handbook. Chloro-
phylls and Bilins: Biosynthesis Structure and Degradation (Kadish,
K. M., Smith, K. M., and Guilard, R., Eds.) pp 211-235, Academic
Press, New York.
2. Foresti, R., Green, C. J., and Motterlini, R. (2004) Generation of bile
pigments by haem oxygenase: A refined cellular strategy in response
to stressful insults. Biochem. Soc. Symp., 177–192.
3. Wilks, A. (2002) Heme oxygenase: Evolution, structure, and mecha-
nism. Antioxid. Redox Signaling 4, 603–614.
4. McDonagh, A. F. (2001) Turning green to gold. Nat. Struct. Biol. 8,
198–200.
5. Kapitulnik, J., and Maines, M. D. (2009) Pleiotropic functions of
biliverdin reductase: Cellular signaling and generation of cytoprotec-
tive and cytotoxic bilirubin. Trends Pharmacol. Sci. 30, 129–137.
6. Frankenberg, N., Mukougawa, K., Kohchi, T., and Lagarias,
J. C. (2001) Functional genomic analysis of the HY2 family of

Article
Biochemistry, Vol. 49, No. 29, 2010 6081
ferredoxin-dependent bilin reductases from oxygenic photosynthetic
organisms. Plant Cell 13, 965–978.
7. Dammeyer, T., and Frankenberg-Dinkel, N. (2008) Function and
distribution of bilin biosynthesis enzymes in photosynthetic orga-
nisms. Photochem. Photobiol. Sci. 7, 1121–1130.
Hildebrandt, P., and Vierstra, R. D. (2009) Cyanochromes are blue/
green light photoreversible photoreceptors defined by a stable double
cysteine linkage to a phycoviolobilin-type chromophore. J. Biol.
Chem. 284, 29757–29772.
32. Hahn, J., Strauss, H. M., Landgraf, F. T., Gimenez, H. F., Lochnit,
G., Schmieder, P., and Hughes, J. (2006) Probing protein-chromophore
interactions in Cph1 phytochrome by mutagenesis. FEBS J. 273,
1415–1429.
8. Wegele, R., Tasler, R., Zeng, Y., Rivera, M., and Frankenberg-
Dinkel, N. (2004) The heme oxygenase(s)-phytochrome system of
Pseudomonas aeruginosa. J. Biol. Chem. 279, 45791–45802.
9. Montgomery, B. L., and Lagarias, J. C. (2002) Phytochrome ancestry.
Sensors of bilins and light. Trends Plant Sci. 7, 357–366.
10. Karniol, B., Wagner, J. R., Walker, J. M., and Vierstra, R. D.
(2005) Phylogenetic analysis of the phytochrome superfamily re-
veals distinct microbial subfamilies of photoreceptors. Biochem. J.
392, 103–116.
11. Lamola, A. A., Blumberg, W. E., McClead, R., and Fanaroff, A.
(1981) Photoisomerized bilirubin in blood from infants receiving
phototherapy. Proc. Natl. Acad. Sci. U.S.A. 78, 1882–1886.
12. McDonagh, A. F., Palms, L. A., Trull, F. R., and Lightner, D. A.
(1982) Phototherapy for neonatal jaundice. Configurational isomers
of bilirubin. J. Am. Chem. Soc. 104, 6865–6869.
33. von Stetten, D., Seibeck, S., Michael, N., Scheerer, P., Mroginski,
M. A., Murgida, D. H., Krauss, N., Heyn, M. P., Hildebrandt, P.,
Borucki, B., and Lamparter, T. (2007) Highly conserved residues Asp-
197 and His-250 in Agp1 phytochrome control the proton affinity of
the chromophore and Pfr formation. J. Biol. Chem. 282, 2116–2123.
34. Yang, X., Stojkovic, E. A., Kuk, J., and Moffat, K. (2007) Crystal
structure of the chromophore binding domain of an unusual bacterio-
phytochrome, RpBphP3, reveals residues that modulate photocon-
version. Proc. Natl. Acad. Sci. U.S.A. 104, 12571–12576.
35. Ikeuchi, M., and Ishizuka, T. (2008) Cyanobacteriochromes: A new
superfamily of tetrapyrrole-binding photoreceptors in cyanobacteria.
Photochem. Photobiol. Sci. 7, 1159–1167.
36. Hanzawa, H., Inomata, K., Kinoshita, H., Kakiuchi, T., Jayasundera,
K. P., Sawamoto, D., Ohta, A., Uchida, K., Wada, K., and Furuya, M.
(2001) In vitro assembly of phytochrome B apoprotein with synthetic
analogs of the phytochrome chromophore. Proc. Natl. Acad. Sci. U.S.A.
98, 3612–3617.
37. Hanzawa, H., Shinomura, T., Inomata, K., Kakiuchi, T., Kinoshita,
H., Wada, K., and Furuya, M. (2002) Structural requirement of bilin
chromophore for the photosensory specificity of phytochromes A and
B. Proc. Natl. Acad. Sci. U.S.A. 99, 4725–4729.
13. Zunszain, P. A., Ghuman, J., McDonagh, A. F., and Curry, S. (2008)
Crystallographic analysis of human serum albumin complexed with
4Z,15E-bilirubin-IXR. J. Mol. Biol. 381, 394–406.
14. Kapitulnik, J. (2004) Bilirubin: An endogenous product of heme
degradation with both cytotoxic and cytoprotective properties. Mol.
Pharmacol. 66, 773–779.
15. Glazer, A. N. (1988) Phycobiliproteins. Methods Enzymol. 167,
291–303.
16. Scheer, H., and Zhao, K. H. (2008) Biliprotein maturation: The
chromophore attachment. Mol. Microbiol. 68, 263–276.
17. Rockwell, N. C., Su, Y. S., and Lagarias, J. C. (2006) Phytochrome
structure and signaling mechanisms. Annu. Rev. Plant Biol. 57, 837–858.
18. Giraud, E., and Vermeglio, A. (2008) Bacteriophytochromes in
anoxygenic photosynthetic bacteria. Photosynth. Res. 97, 141–153.
19. Rockwell, N. C., and Lagarias, J. C. (2010) A Brief History of
Phytochromes. ChemPhysChem 11, 1172–1180.
38. Inomata, K. (2008) Studies on the structure and function of phyto-
chromes as photoreceptors based on synthetic organic chemistry.
Bull. Chem. Soc. Jpn. 81, 25–59.
€
39. Bongards, C., and Gartner, W. (2010) The Role of the Chromo-
phore in the Biological Photoreceptor Phytochrome: An Approach
Using Chemically Synthesized Tetrapyrroles. Acc. Chem. Res. 43,
485–495.
20. Scheerer, P., Michael, N., Park, J. H., Nagano, S., Choe, H. W.,
Inomata, K., Borucki, B., Krauss, N., and Lamparter, T. (2010)
Light-induced conformational changes of the chromophore and the
protein in phytochromes: Bacterial phytochromes as model systems.
ChemPhysChem 11, 1090–1105.
40. Elich, T. D., and Lagarias, J. C. (1989) Formation of a photorever-
sible phycocyanobilin-apophytochrome adduct in vitro. J. Biol. Chem.
264, 12902–12908.
41. Elich, T. D., McDonagh, A. F., Palma, L. A., and Lagarias, J. C.
(1989) Phytochrome chromophore biosynthesis. Treatment of tetra-
pyrrole-deficient Avena explants with natural and non-natural bila-
trienes leads to formation of spectrally active holoproteins. J. Biol.
Chem. 264, 183–189.
42. Li, L., and Lagarias, J. C. (1992) Phytochrome assembly: Defining
chromophore structural requirements for covalent attachment and
photoreversibility. J. Biol. Chem. 267, 19204–19210.
43. Bhoo, S. H., Hirano, T., Jeong, H. Y., Lee, J. G., Furuya, M., and
Song, P. S. (1997) Phytochrome photochromism probed by site-
directed mutations and chromophore esterification. J. Am. Chem.
Soc. 119, 11717–11718.
21. Murphy, J. T., and Lagarias, J. C. (1997) The Phytofluors: A new class
of fluorescent protein probes. Curr. Biol. 7, 870–876.
22. Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002)
A light-switchable gene promoter system. Nat. Biotechnol. 20, 1041–1044.
23. Fischer, A. J., and Lagarias, J. C. (2004) Harnessing phytochrome’s
glowing potential. Proc. Natl. Acad. Sci. U.S.A. 101, 17334–17339.
24. Su, Y. S., and Lagarias, J. C. (2007) Light independent phytochrome
signaling mediated by dominant GAF-domain tyrosine mutants of
Arabidopsis phytochromes in transgenic plants. Plant Cell 19, 2124–
2139.
25. Leung, D. W., Otomo, C., Chory, J., and Rosen, M. K. (2008)
Genetically encoded photoswitching of actin assembly through the
Cdc42-WASP-Arp2/3 complex pathway. Proc. Natl. Acad. Sci. U.S.A.
105, 12797–12802.
26. Shu, X., Royant, A., Lin, M. Z., Aguilera, T. A., Lev-Ram, V.,
Steinbach, P. A., and Tsien, R. Y. (2009) Mammalian expression of
infrared fluorescent proteins engineered from a bacterial phyto-
chrome. Science 324, 804–807.
44. Kami, C., Mukougawa, K., Muramoto, T., Yokota, A., Shinomura,
T., Lagarias, J. C., and Kohchi, T. (2004) Complementation of
phytochrome chromophore-deficient Arabidopsis by expression of
phycocyanobilin:ferredoxin oxidoreductase. Proc. Natl. Acad. Sci.
U.S.A. 101, 1099–1104.
45. Rockwell, N. C., Shang, L., Martin, S. S., and Lagarias, J. C. (2009)
Distinct classes of red/far-red photochemistry within the phyto-
chrome superfamily. Proc. Natl. Acad. Sci. U.S.A. 106, 6123–6127.
46. Ma, J. S., and Lightner, D. A. (1984) Facile Preparation of Symmetric
Bilirubins IIIR and XIIIR from IXR. J. Heterocycl. Chem. 21, 1005–
1008.
27. Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009)
Spatiotemporal control of cell signalling using a light-switchable
protein interaction. Nature 461, 997–1001.
28. Fischer, A. J., Rockwell, N. C., Jang, A. Y., Ernst, L. A., Waggoner,
A. S., Duan, Y., Lei, H., and Lagarias, J. C. (2005) Multiple roles of a
conserved GAF domain tyrosine residue in cyanobacterial and plant
phytochromes. Biochemistry 44, 15203–15215.
29. Rockwell, N. C., Njuguna, S. L., Roberts, L., Castillo, E., Parson,
V. L., Dwojak, S., Lagarias, J. C., and Spiller, S. C. (2008) A second
conserved GAF domain cysteine is required for the blue/green photo-
reversibility of cyanobacteriochrome Tlr0924 from Thermosynecho-
coccus elongatus. Biochemistry 47, 7304–7316.
30. Wagner, J. R., Zhang, J., von Stetten, D., Gunther, M., Murgida,
D. H., Mroginski, M. A., Walker, J. M., Forest, K. T., Hildebrandt,
P., and Vierstra, R. D. (2008) Mutational analysis of Deinococcus
radiodurans bacteriophytochrome reveals key amino acids necessary
for the photochromicity and proton exchange cycle of phytochromes.
J. Biol. Chem. 283, 12212–12226.
47. McDonagh, A. F. (1979) Bile Pigments: Bilatrienes and 5,15-Bila-
dienes. In The Porphyrins (Dolphin, D., Ed.) pp 293-491, Academic
Press, New York.
48. Smith, P. K., Krohn, R. I., Hemanson, G. T., Mallia, A. K., Gartner,
F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olsen,
B. J., and Klenk, D. C. (1985) Measurement of Protein using
Bicinchoninic Acid. Anal. Biochem. 150, 76–85.
49. Bradford, M. M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein using the principle
of protein-dye binding. Anal. Biochem. 72, 248–254.
€
50. Lindner, I., Knipp, B., Braslavsky, S. E., Gartner, W., and Schaffner,
K. (1998) A novel chromophore selectively modifies the spectral
properties of one of the two stable states of the plant photoreceptor
phytochrome. Angew. Chem., Int. Ed. 37, 1843–1846.
51. Frankenberg, N., and Lagarias, J. C. (2003) Phycocyanobilin:ferre-
doxin oxidoreductase. Biochemical and spectroscopic characteriza-
tion. J. Biol. Chem. 278, 9219–9226.
31. Ulijasz, A. T., Cornilescu, G., von Stetten, D., Cornilescu, C.,
Velazquez Escobar, F., Zhang, J., Stankey, R. J., Rivera, M.,

6082 Biochemistry, Vol. 49, No. 29, 2010
Shang et al.
52. Tu, S.-L., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C.
(2004) Biliverdin reduction by cyanobacterial phycocyanobilin:ferre-
doxin oxidoreductase (PcyA) proceeds via linear tetrapyrrole radical
intermediates. J. Am. Chem. Soc. 126, 8682–8693.
53. Schluchter, W. M. (1994) The Characterization of Photosystem I and
Ferredoxin-NADPþ Oxidoreductase in the Cyanobacterium Syne-
chococcus sp. PCC 7002. Ph.D. Thesis, p 300, Department of Bio-
chemistry and Molecular Biology, Pennsylvania State University,
University Park, PA.
54. Whitby, F. G., Phillips, J. D., Hill, C. P., McCoubrey, W., and
Maines, M. D. (2002) Crystal structure of a biliverdin IXR reductase
enzyme-cofactor complex. J. Mol. Biol. 319, 1199–1210.
55. Murphy, J. T., and Lagarias, J. C. (1997) Purification and characte-
rization of recombinant affinity peptide-tagged oat phytochrome A.
Photochem. Photobiol. 65, 750–758.
56. McDonagh, A. F., and Assisi, F. (1972) Direct evidence for the acid-
catalysed isomeric scrambling of bilirubin IXR. J. Chem. Soc., Chem.
Commun., 117–119.
57. Lightner, D. A., Holmes, D. L., and McDonagh, A. F. (1996) On the
Acid Dissociation Constants of Bilirubin and Biliverdin: pKa Values
from C-13 NMR Spectroscopy. J. Biol. Chem. 271, 2397–2405.
58. Maines, M. D. (2005) New insights into biliverdin reductase functions:
Linking heme metabolism to cell signaling. Physiology 20, 382–389.
59. Schluchter, W. M., and Glazer, A. N. (1997) Characterization of
cyanobacterial biliverdin reductase: Conversion of biliverdin to bilir-
ubin is important for normal phycobiliprotein biosynthesis. J. Biol.
Chem. 272, 13562–13569.
60. Franklin, E., Browne, S., Hayes, J., Boland, C., Dunne, A., Elliot, G.,
and Mantle, T. J. (2007) Activation of biliverdin-IXR reductase by
inorganic phosphate and related anions. Biochem. J. 405, 61–67.
61. Hayes, J. M., and Mantle, T. J. (2009) The effect of pH on the
initial rate kinetics of the dimeric biliverdin-IXalpha reductase from the
cyanobacterium Synechocystis PCC6803. FEBS J. 276, 4414–4425.
62. Fakhrai, H., and Maines, M. D. (1992) Expression and characteriza-
tion of a cDNA for rat kidney biliverdin reductase. J. Biol. Chem. 267,
4023–4029.
63. Hagiwara, Y., Sugishima, M., Khawn, H., Kinoshita, H., Inomata,
K., Shang, L., Lagarias, J. C., Takahashi, Y., and Fukuyama, K.
(2010) Structural insights into vinyl reduction regiospecificity of
phycocyanobilin:ferredoxin oxidoreductase (PcyA). J. Biol. Chem.
285, 1000–1007.
64. Hagiwara, Y., Sugishima, M., Takahashi, Y., and Fukuyama, K.
(2006) Crystal structure of phycocyanobilin:ferredoxin oxidoreduc-
tase in complex with biliverdin IXR, a key enzyme in the biosynthesis
of phycocyanobilin. Proc. Natl. Acad. Sci. U.S.A. 103, 27–32.
65. Hagiwara, Y., Sugishima, M., Takahashi, Y., and Fukuyama, K.
(2006) Induced-fitting and electrostatic potential change of PcyA
upon substrate binding demonstrated by the crystal structure of the
substrate-free form. FEBS Lett. 580, 3823–3828.
66. Tu, S. L., Fisher, A. J., Rockwell, N. C., and Lagarias, J. C. (2007)
Insight into the radical mechanism of phycocyanobilin:ferredoxin
oxidoreductase (PcyA) revealed by X-ray crystallography and bio-
chemical analysis. Biochemistry 46, 1484–1494.
67. Borucki, B., Otto, H., Rottwinkel, G., Hughes, J., Heyn, M. P., and
Lamparter, T. (2003) Mechanism of Cph1 phytochrome assembly
from stopped-flow kinetics and circular dichroism. Biochemistry 42,
13684–13697.
68. Essen, L. O., Mailliet, J., and Hughes, J. (2008) The structure of a
complete phytochrome sensory module in the Pr ground state. Proc.
Natl. Acad. Sci. U.S.A. 105, 14709–14714.
69. Goller, A. H., Strehlow, D., and Hermann, G. (2005) The excited-
state chemistry of phycocyanobilin: A semiempirical study. Chem-
PhysChem 6, 1259–1268.
70. Wagner, J. R., Zhang, J., Brunzelle, J. S., Vierstra, R. D., and Forest,
K. T. (2007) High resolution structure of Deinococcus bacteriophy-
tochrome yields new insights into phytochrome architecture and
evolution. J. Biol. Chem. 282, 12298–12309.
71. Spillane, K. M., Dasgupta, J., Lagarias, J. C., and Mathies, R. A.
(2009) Homogeneity of phytochrome Cph1 vibronic absorption
revealed by resonance Raman intensity analysis. J. Am. Chem. Soc.
131, 13946–13948.
74. Zhao, K. H., and Scheer, H. (1995) Type I and Type II Reversible
Photochemistry of Phycoerythrocyanin R-Subunit from Mastigocla-
dus laminosus Both Involve Z, E Isomerization of Phycoviolobilin
Chromophore and Are Controlled by Sulfhydryls in Apoprotein.
Biochim. Biophys. Acta 1228, 244–253.
75. Borucki, B., Seibeck, S., Heyn, M. P., and Lamparter, T. (2009)
Characterization of the covalent and noncovalent adducts of Agp1
phytochrome assembled with biliverdin and phycocyanobilin by
circular dichroism and flash photolysis. Biochemistry 48, 6305–6317.
76. Stoll, S., Gunn, A., Brynda, M., Sughrue, W., Kohler, A. C., Ozar-
owski, A., Fisher, A. J., Lagarias, J. C., and Britt, R. D. (2009)
Structure of the biliverdin radical intermediate in phycocyanobilin:
ferredoxin oxidoreductase identified by high-field EPR and DFT.
J. Am. Chem. Soc. 131, 1986–1995.
77. Nogales, D., and Lightner, D. A. (1995) On the structure of bilirubin
in solution. 13C[1H] heteronuclear Overhauser effect NMR analyses in
aqueous buffer and organic solvents. J. Biol. Chem. 270, 73–77.
78. Awruch, J., Tomaro, M. L., Frydman, R. B., and Frydman, B. (1984)
The specificity of biliverdin reductase. The reduction of biliverdin
XIII isomers. Biochim. Biophys. Acta 787, 146–151.
79. Tomaro, M. L., Frydman, R. B., Awruch, J., Valasinas, A., Frydman,
B., Pandey, R. K., and Smith, K. M. (1984) The specificity of
biliverdin reductase. A study with different biliverdin types. Biochim.
Biophys. Acta 791, 350–356.
80. Frydman, R. B., Tomaro, M. L., Rosenfeld, J., Awruch, J., Sambrotta,
L., Valasinas, A., and Frydman, B. (1987) Biliverdin reductase: Substrate
specificity and kinetics. Biochim. Biophys. Acta 916, 500–511.
81. Frydman, R. B., Bari, S., Tomaro, M. L., and Frydman, B. (1990) The
Enzymatic and Chemical Reduction of Extended Biliverdins. Bio-
chem. Biophys. Res. Commun. 171, 465–473.
82. Terry, M. J., Maines, M. D., and Lagarias, J. C. (1993) Inactivation of
Phytochrome-Chromophore and Phycobiliprotein-Chromophore
Precursors by Rat Liver Biliverdin Reductase. J. Biol. Chem. 268,
26099–26106.
83. Cunningham, O., Dunne, A., Sabido, P., Lightner, D., and Mantle,
T. J. (2000) Studies on the specificity of the tetrapyrrole substrate for
human biliverdin-IX R reductase and biliverdin-IX β reductase:
Structure-activity relationships define models for both active sites.
J. Biol. Chem. 275, 19009–19017.
84. Franklin, E. M., Browne, S., Horan, A. M., Inomata, K., Hammam,
M. A., Kinoshita, H., Lamparter, T., Golfis, G., and Mantle, T. J.
(2009) The use of synthetic linear tetrapyrroles to probe the verdin
sites of human biliverdin-IXR reductase and human biliverdin-IXβ
reductase. FEBS J. 276, 4405–4413.
85. Litts, J. C., Kelly, J. M., and Lagarias, J. C. (1983) Structure-function
studies on phytochrome. Preliminary characterization of highly puri-
fied phytochrome from Avena sativa enriched in the 124-kilodalton
species. J. Biol. Chem. 258, 11025–11031.
86. Yang, X., Kuk, J., and Moffat, K. (2008) Crystal structure of
Pseudomonas aeruginosa bacteriophytochrome: Photoconversion
and signal transduction. Proc. Natl. Acad. Sci. U.S.A. 105, 14715–
14720.
87. Yang, X., Kuk, J., and Moffat, K. (2009) Conformational differences
between the Pfr and Pr states in Pseudomonas aeruginosa bacterio-
phytochrome. Proc. Natl. Acad. Sci. U.S.A. 106, 15639–15644.
88. Dasgupta, J., Frontiera, R. R., Taylor, K. C., Lagarias, J. C.and
Mathies, R. A. (2009) Ultrafast excited-state isomerization in phytochrome
revealed by femtosecond stimulated Raman spectroscopy. Proc. Natl.
Acad. Sci. U.S.A. 106, 1784–1789.
89. Mroginski, M. A., von Stetten, D., Escobar, F. V., Strauss, H. M.,
Kaminski, S., Scheerer, P., Gunther, M., Murgida, D. H., Schmieder,
P., Bongards, C., Gartner, W., Mailliet, J., Hughes, J., Essen, L. O.,
and Hildebrandt, P. (2009) Chromophore structure of cyanobac-
terial phytochrome Cph1 in the Pr state: Reconciling structural and
spectroscopic data by QM/MM calculations. Biophys. J. 96, 4153–
4163.
90. Rohmer, T., Lang, C., Hughes, J., Essen, L. O., Gartner, W., and
Matysik, J. (2008) Light-induced chromophore activity and signal
transduction in phytochromes observed by 13C and 15N magic-angle
spinning NMR. Proc. Natl. Acad. Sci. U.S.A. 105, 15229–15234.
91. Strauss, H. M., Hughes, J., and Schmieder, P. (2005) Heteronuclear
solution-state NMR studies of the chromophore in cyanobacterial
phytochrome Cph1. Biochemistry 44, 8244–8250.
92. Ulijasz, A. T., Cornilescu, G., Cornilescu, C. C., Zhang, J., Rivera,
M., Markley, J. L., and Vierstra, R. D. (2010) Structural basis for the
photoconversion of a phytochrome to the activated Pfr form. Nature
463, 250–254.
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72. Mimuro, M., Fuglistaller, P., Rumbeli, R., and Zuber, H. (1986)
Functional assignment of chromophores and energy transfer in C
phycocyanin isolated from the thermophilic cyanobacterium Masti-
gocladus laminosus. Biochim. Biophys. Acta 848, 155–166.
73. Zhao, K. H., Haessner, R., Cmiel, E., and Scheer, H. (1995) Type I
Reversible Photochemistry of Phycoerythrocyanin Involves Z/E-Iso-
merization of R-84 Phycoviolobilin Chromophore. Biochim. Biophys.
Acta 1228, 235–243.
93. Hughes, J. (2010) Phytochrome three-dimensional structures and
functions. Biochem. Soc. Trans. 38, 710–716.