From the Section of Molecular and Cellular Biology,
University of California, Davis, California 95616
Received for publication, November 14, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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In cyanobacteria, the biosynthesis of the
phycobiliprotein and phytochrome chromophore precursor
phycocyanobilin is catalyzed by the ferredoxin-dependent
enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA),
which mediates an atypical four-electron reduction of biliverdin IX Phycocyanobilin (PCB)1
is a linear tetrapyrrole (bilin) found in cyanobacteria, algae, and
cryptomonads that functions as the direct precursor of the chromophores
of the light-harvesting phycobiliproteins and cyanobacterial/algal
phytochromes (1, 2). The PCB biosynthetic pathway shares common
intermediates with those of heme and chlorophyll to the level of
protoporphyrin IX, whereupon the pathways diverge upon metalation with
either iron or magnesium (1). All phycobilins share the common
intermediacy of biliverdin IX We recently documented that pcyA genes from cyanobacteria
and oxyphotobacteria encode bilin reductases, which catalyze the ferredoxin-dependent reduction of BV IX.
Here we describe the expression, affinity purification, and biochemical
characterization of recombinant PcyA from Anabaena sp. PCC
7120. A monomeric protein with a native Mr of
30,400 ± 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its substrate biliverdin IX
. The enzyme
exhibits a strong preference for plant type [2Fe-2S] ferredoxins; however, flavodoxin can also serve as an electron donor. HPLC analyses
establish that catalysis proceeds via the two electron-reduced intermediate 181,182-dihydrobiliverdin,
indicating that exovinyl reduction precedes A-ring (endovinyl)
reduction. Substrate specificity studies indicate that the arrangement
of the A- and D-ring substituents alters the positioning of the bilin
substrate within the enzyme, profoundly influencing the course of
catalysis. Based on these observations and the apparent lack of a metal
or small molecule cofactor, a radical mechanism for biliverdin IX
reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(BV IX
), which is derived from heme
(1, 3). BV IX
is the product of heme oxygenase-mediated cleavage of
the heme macrocycle, which yields equimolar quantities of BV IX
, CO,
and iron. The metabolic fate of BV IX
differs in mammals,
cyanobacteria, and plants, where BV IX
is metabolized by different
reductases with unique double bond specificities. In contrast with the
mammalian NAD(P)H-dependent BV IX
reductase,
cyanobacteria and red algae possess ferredoxin-dependent bilin reductases primarily for the synthesis of the linear tetrapyrrole precursors of their phycobiliprotein light-harvesting antennae complexes, whereas evolutionarily related
ferredoxin-dependent bilin reductases are found in higher
plants for the synthesis of the phytochrome chromophore precursor
phytochromobilin (P
B) (3, 4).
to
(3Z)-PCB, the bilin precursor of their
phycobiliprotein and phytochrome chromophores. Designated
phycocyanobilin:ferredoxin oxidoreductases (EC 1.3.7.5), PcyA enzymes
are atypical bilin reductases, because they catalyze a four-electron
reduction; all others catalyze two-electron reductions (4). Formally
two-electron reductions of vinyl substituents on the pyrrole A- and
D-rings of BV IX
, the sequence of reductions mediated by
PcyA, is presently unknown (see Fig. 1). Neither of the two
likely dihydrobiliverdin (DHBV) intermediates, (3Z)-P
B or
181,182-DHBV IX
, have yet been detected (4).
Since (3Z)-P
B is an intermediate in the biosynthesis of
PCB, the precursor of the phytochrome chromophore in the green alga
Mesotaenium caldariorum (5), its intermediacy in the
PcyA-mediated biosynthesis of PCB in cyanobacteria is therefore a
reasonable possibility. Previous studies have shown that the
biosynthesis of PCB in the red alga Cyanidium caldarium
proceeds via phycoerythrobilin as an intermediate (Fig.
1) (1). The enzyme(s) that catalyze the
conversion of phycoerythrobilin to PCB have not yet been identified;
hence, it is conceivable that PcyA might also mediate this
conversion.
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Fig. 1.
The reduction of biliverdin
IX by PcyA can proceed via two possible
intermediates. PcyA-mediated reduction of BV IX
could result in
the formation of (3Z)-P
B or
181,182-dihydrobiliverdin IX
as an
intermediate. Results presented here support the intermediacy of
181,182-dihydrobiliverdin IX
; however,
(3Z)-P
B is also metabolized by PcyA (see
"Results" for details). An alternative pathway for the
formation of PCB in the red alga C. caldarium via the
intermediacy of 15,16-dihydrobiliverdin IX
and phycoerythrobilin is
also shown in the center. This conversion is catalyzed by the bilin
reductases, PebA and PebB, and an as yet unidentified isomerase (1,
4).
This study was undertaken to characterize the biochemical properties of
a representative PcyA enzyme from the filamentous cyanobacterium
Anabaena sp. PCC 7120. The specific objectives of these
experiments were to identify the semireduced intermediate produced
during the catalysis of BV and to probe the bilin substrate specificity
of this unusual ferredoxin-dependent four-electron reductase. Based on these investigations, a chemical mechanism for
PcyA-mediated bilin reduction is proposed.
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EXPERIMENTAL PROCEDURES |
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Reagents-- Unless otherwise specified, all chemical reagents were American Chemical Society grade or better. Glutathione-agarose, spinach ferredoxin, Clostridium pasteurianum ferredoxin, ferredoxin:NADP+ oxidoreductase (FNR), and size exclusion molecular weight markers (MW-GF-200) were purchased from Sigma. Restriction enzymes and Taq polymerase were obtained from Invitrogen. HPLC grade acetone, chloroform, and 80% formic acid were purchased from Fisher. The expression vector pGEX-6P-1 and PreScissionTM protease were obtained from Amersham Biosciences. Centricon-10 concentrator devices were purchased from Amicon (Beverly, MA).
Bilin Preparations--
BV IX, BV XIII
, BV III
, PCB,
and P
B preparations used as substrate and/or HPLC standards were
obtained as described previously (6, 7).
181,182-DHBV IX
was synthesized by acid
scrambling of a mixture of meso-BR III
and BR XIII
followed by
oxidation with 2,3-dichloro-5,6-dicyanobenzoquinone (8). Meso-BR III
was kindly provided by Dr. D. A. Lightner (University of Nevada,
Reno, NV). BR XIII
was prepared by acid scrambling of commercially
obtained BR (7, 9).
Expression and Purification of PcyA--
Anabaena sp.
PCC 7120 pcyA was cloned into the Escherichia
coli expression vector pGEX-6-P1 (Amersham Biosciences) to produce pGEXpcyA (4). E. coli strain DH5 containing
pGEXpcyA was induced to express glutathione
S-transferase-PcyA, which was purified according to
instructions supplied by the manufacturer and protocols described
earlier (10). Proteolytic cleavage with the PreScissionTM protease
yielded the native protein with the N-terminal amino acid extension
GPLGSPEF and with the initiator methionine residue changed to
isoleucine. Purified PcyA protein concentration was estimated from the
absorbance at 280 nm using the calculated
280 nm of
29,726 M
1 cm
1 (11).
Purification of Recombinant
Reductants--
Synechococcus sp. PCC 7002 ferredoxin and
flavodoxin clones, obtained from Dr. D. A. Bryant, were expressed
and purified as described previously (12, 13). Expression and
purification of putidaredoxin and putidaredoxin reductase, whose clones
were kindly provided by Dr. Paul Ortiz de Montellano (University of California, San Francisco), were performed as
described.2 Flavodoxin was
quantified by absorption at 467 nm and an absorption coefficient of
467 nm 9,500 M
1
cm
1 (13), whereas putidaredoxin and putidaredoxin
reductase were quantified by absorption at 454/415 nm, respectively,
using the absorption coefficients of
454 nm 10,000 M
1 cm
1 and
415 nm 11,100 M
1
cm
1.2
Standard Bilin Reductase Activity Assay--
Assays for bilin
reductase activity were performed as described previously (10, 14).
Standard assays contained 1.5 µM PcyA, 4.8 µM ferredoxin, and 5 µM BV IX in 25 mM TES-KOH, pH 7.5 (assay buffer), and were incubated for
30 min at 28 °C under green safe light unless otherwise specified.
Following catalysis, bilins were isolated using a C18 Sep-Pak
column (Waters) and evaporated to dryness in vacuo
(4).
Direct HPLC Analysis-- Bilin reaction products were dissolved in 10 µl of Me2SO and diluted with 200 µl of the HPLC mobile phase. Following brief centrifugation and filtration through a 0.45-µm polytetrafluoroethylene syringe filter, bilins were resolved by reversed phase chromatography using an Agilent Technologies 1100 Liquid Chromatograph. The HPLC column used for all of the analyses was a 4.6 × 250-mm Phenomenex Ultracarb 5-µm ODS(20) analytical column with a 4.6 × 30-mm guard column of the same material. The mobile phase consisted of acetone, 20 mM formic acid (50:50 by volume), and the flow rate was 0.6 ml/min. Eluates were monitored at 650, 560, and 380 nm using an Agilent Technologies 1100 series diode array detector. As needed, complete spectra were obtained for the peaks desired. Peak areas were quantitated using Agilent Technologies Chemstation software.
Size Exclusion Chromatography--
An Amersham
Biosciences Superdex 200 HR10/30 size exclusion column was
equilibrated in 50 mM TES-KOH buffer, pH 7.5, containing 100 mM KCl and 10% (v/v) glycerol (size exclusion
chromatography buffer) at a flow rate of 0.4 ml/min. Standards with
known Mr (i.e. -amylase, 200,000;
alcohol dehydrogenase, 150,000; bovine serum albumin, 66,000; carbonic
anhydrase, 29,000; cytochrome c, 12,600) were applied to the
column (100 µg), and their elution volumes were determined
spectroscopically. Anabaena sp. PcyA, PcyA/BV (1:1,
mol/mol), Fd/PcyA (2:1, mol/mol), and Fd/PcyA/BV (2:1:1, mol/mol/mol)
were chromatographed under identical conditions.
Glycerol Gradient Centrifugation-- PcyA preparations (40 µg) were sedimented through a 2.5-ml continuous 10-25% glycerol gradient in size exclusion chromatography buffer. A detailed experimental procedure described previously was used for sedimentation coefficient determination (15).
Spectroscopic Analysis of Biliverdin Binding--
Increasing
amounts of PcyA were added to 5 µM (final concentration)
BV IX, BV XIII
, or BV III
solutions in a final volume of 500 µl of 25 mM TES-KOH, pH 7.5, buffer under green safe
light. After incubation for 30 min at room temperature, absorbance
spectra were recorded using an HP 8453 spectrophotometer. Normalization of the spectra and spectral deconvolution were performed using Microsoft Excel. To obtain bilin-PcyA dissociation constants, absorbance differences (
A) at the
max of
each bilin-PcyA complex were plotted as a function of PcyA
concentration. Dissociation constants were obtained by fitting this
data to the hyperbolic equation
A =
Amax × [PcyA]/(Kapp + [PcyA]) using DeltaGraph® Pro version 3.5 (DeltaPoint, Monterey,
CA), where Kd = Kapp
2.5
µM.
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RESULTS |
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Expression and Purification of Recombinant
Phycocyanobilin:Ferredoxin Oxidoreductase--
The Anabaena
sp. PCC 7120 pcyA gene was expressed using a tac
promoter-driven N-terminal glutathione S-transferase fusion expression system. Recombinant PcyA, obtained by "on-column"
proteolytic cleavage of the glutathione S-transferase fusion
protein, was purified to ~90% homogeneity as shown in Fig.
2A. On-column cleavage was preferable to "in-solution" proteolysis of the
glutathione S-transferase-PcyA fusion protein, which led to
extensive protein precipitation and poor protein recovery. One-liter
bacterial cultures typically yielded 3 mg of on-column cleaved PcyA.
All results presented here correspond to PcyA. However, glutathione
S-transferase-PcyA fusion protein preparations showed nearly
identical catalytic properties (data not shown).
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Determination of the Native Molecular Mass of PcyA--
The native
molecular mass of PcyA was determined using size exclusion
chromatography and glycerol gradient sedimentation (Fig. 2,
B and C). A relative molecular weight of
30,400 ± 5,000 for PcyA was deduced with both methods, which is
in good agreement with the calculated molecular mass of 28,726 daltons.
Thus, recombinant PcyA appears to be a monomeric enzyme. In order to
determine whether PcyA can form a stable complex with spinach
ferredoxin, PcyA was incubated with a 2-fold molar excess of
spinach Fd and evaluated by both methods. Higher order complex
formation between PcyA and Fd was not observed with either method under
the conditions examined; nor did the addition of a 2-fold molar excess
of BV IX influence the result (data not shown).
PcyA Lacks Metal or Small Molecule Cofactors-- Purified recombinant PcyA was analyzed using absorption spectroscopy for the presence of light-absorbing cofactors such as hemes, flavins, iron-sulfur clusters, etc. Spectroscopic evidence for any of these cofactors was not obtained for PcyA at concentrations as high as 5 mg/ml. In order to understand whether solvent-accessible metal ions are critical for activity (directly or indirectly as structural components), purified PcyA was incubated with the metal chelators EDTA (10 mM), 1,10-phenanthroline, and 2,2'-dipyridyl (5 mM each). After removal of the chelator from the protein by passing the mixture through a G-25 desalting column (Amersham Biosciences), enzyme activity was determined. None of the chelators had any inhibitory effect on the activity of PcyA (data not shown).
Reductant Specificity of PcyA--
The enzymes that mediate the
reductive conversion of BV IX to phycobilins are all dependent on
plant type [2Fe-2S] ferredoxins (4, 14). For this reason, all PcyA
assays were performed in the presence of saturating levels of spinach
ferredoxin and the ferredoxin-reducing system, consisting of spinach
FNR and NADPH. Omission of any of these components led to no PcyA
activity (data not shown) (4). To test whether other reductants can serve as electron donors to PcyA, we tested recombinant
Synechococcus sp. PCC 7002 ferredoxin and flavodoxin also in
combination with spinach FNR. Other reductants tested included C. pasteurianum ferredoxin, a 2[4Fe-4S] ferredoxin, and a
putidaredoxin/putidaredoxin reductase system from Pseudomonas
putida. As shown in Table I, maximum
PcyA activity was obtained with plant type ferredoxin either from
spinach or Synechococcus, whereas the FMN-containing redox
protein flavodoxin less effectively supported PcyA catalysis. C. pasteurianum ferredoxin and the putidaredoxin/putidaredoxin reductase system from P. putida were considerably less
active.
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Bilin Binding to PcyA--
Bilin binding experiments demonstrated
that PcyA forms a complex with its bilin substrate that is stable
through ultrafiltration, size exclusion chromatography, and dialysis.
Spectrophotometric titration experiments with its natural substrate BV
IX and the two analogs, BV XIII
and BV III
, are shown in Fig.
3A. Upon binding to PcyA,
significant blue shifts of the long wavelength absorption maxima were
detected for all of the bilin analogs along with an increase in molar
absorption coefficient and the appearance of a shoulder at longer
wavelengths. The appearance of the shoulder was evident only for BV
IX
and BV XIII
, but not BV III
(Fig. 3B), a result
that was consistent with the ability of PcyA to metabolize these two
bilins (see below). Fig. 3C depicts replots of these
absorption changes as a function of increasing PcyA concentration. From
hyperbolic curve fitting of this data, the equilibrium dissociation constants were estimated to be 12.5, 14.5, and 4.5 µM for
the respective IX
, XIII
, and III
isomers under these
experimental conditions. As a control, BV IX
binding experiments
were also performed using bovine carbonic anhydrase from the Sigma
MW-GF-200 molecular weight kit. No spectral changes were observed with
increasing carbonic anhydrase concentration, indicating that the
altered spectra reflected the formation of bilin-PcyA complexes (Fig. 3C).
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The PcyA Two-electron Reduced Intermediate--
The
conversion of BV to PCB is a four-electron reduction that formally
consists of sequential two-electron reductions with the intermediacy of
a DHBV. As shown in Fig. 1, the most likely candidates for this
intermediate are (3Z)-PB or
181,182-DHBV IX
, in which initial reduction
occurs at the A- or D-rings of BV, respectively. To identify the
putative DHBV intermediate, time course experiments were performed.
HPLC analyses revealed the transient appearance of a new pigment during
the course of catalysis (Fig.
4 and Table
II). This new pigment, which eluted earlier than (3Z)-PCB (labeled I in Fig.
4A), reached a maximum level within 10 min and disappeared
after 30 min (Fig. 4B). Although pigment I eluted at the
same retention time as (3E)-P
B, its absorption spectrum
differed from that of (3E)-P
B (Table II). The time course of the disappearance of BV IX
, the appearance/disappearance of pigment I, and the appearance of the two isomers of PCB, shown in Fig.
4B, supports the intermediacy of pigment I in the
PcyA-mediated conversion of BV IX
to PCB.
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To verify that pigment I was a bona fide intermediate in the formation
of PCB, it was collected and tested for its ability to bind to apo-Cph1
using a coupled phytochrome assembly assay (10, 14) and for its
ability to be further metabolized by PcyA to PCB using HPLC. As shown
in Fig. 4C, isolated pigment I failed to produce a
photoactive phytochrome upon incubation with apo-Cph1, indicating that
pigment I was not (3E)-PB or
(3Z)/(3E)-PCB. Further incubation of pigment I
with PcyA yielded products that could assemble with apo-Cph1 to produce
a photoactive bilin adduct. Fig. 4C shows that the
phytochrome difference spectrum of this adduct was identical to that of
the authentic PCB adduct, both of which were blue-shifted from that of
the Cph1:P
B adduct. Together with the ability of PcyA to convert
pigment I to a mixture of pigments that co-elute with (3Z)-
and (3E)-PCB (data not shown), these studies support the
conclusion that pigment I is a bona fide
intermediate in the PcyA-mediated conversion of BV to PCB.
181,182-DHBV Is the Intermediate in the
PcyA-mediated Reduction of BV--
Since the intermediate failed to
form a photoactive bilin-adduct with apo-Cph1 but had the same
retention time as (3E)-PB, we tested both
(3E)- and (3Z)-P
B as substrates for PcyA.
These studies showed that (3Z)-P
B, but not
(3E)-P
B, was metabolized by PcyA. (3Z)-P
B
was converted to a mixture of (3Z)- and
(3E)-isomers of PCB, a result that was confirmed by assembly
with apo-Cph1 (data not shown). Since (3Z)-P
B elutes at a
different retention time from the intermediate on the HPLC (Fig.
4A, Table II), these studies confirm that neither P
B
isomer is the semireduced intermediate in the PcyA-mediated reduction
of BV. According to Fig. 1, the other likely intermediate is
181,182-DHBV IX
. For this reason,
181,182-DHBV IX
was synthesized by acid
scrambling of BR XIII
and mBRIII
followed by oxidation as
described under "Experimental Procedures." Fig. 4A shows
that 181,182-DHBV IX
elutes at the same
retention time as pigment I. These studies also show that PcyA converts
181,182-DHBV IX
to a mixture of
(3E)- and (3Z)-PCB, thereby confirming the identity of the semireduced intermediate to be
181,182-DHBV IX
.
Bilin Substrate Specificity Studies--
Since the substrate
analogs BV XIII and BV III
bind to PcyA (see Fig. 3), their
ability to be metabolized by PcyA was also examined. As shown in Fig.
5A, BV XIII
could be
metabolized by PcyA to yield two products. Based on the relative
retention time of known bilins in our HPLC system and the absorbance
spectra of the two products (Fig. 5B), we propose that BV
XIII
is converted by PcyA to the (3E)- and
(3Z)-isomers of iso-P
B (16). This hypothesis is also
supported by the observation that both products yield identical
difference spectra upon incubation with apo-Cph1 (Fig. 5C).
By contrast with the other two BV isomers, BV III
was not
metabolized by PcyA. This result is interesting in view of the
observation that BV III
has the highest binding affinity for PcyA of
the three BV isomers (Fig. 3C). The results of the bilin
substrate specificity experiments are summarized in Table III.
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DISCUSSION |
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PcyA Is a Monomeric Enzyme That Forms a Stable Porphyrin-like
Complex with Bilins--
Among the family of
ferredoxin-dependent bilin reductases, PcyA is
unique in its ability to catalyze the four-electron reduction of BV
IX (4). Like oat phytochromobilin synthase, a
ferredoxin-dependent bilin reductase that converts BV IX
to P
B in plants (14), PcyA is a monomeric enzyme. BV binding neither
promoted PcyA dimerization nor oligomerization, suggesting that the
distinct spectral properties of the three PcyA-BV complexes
studied here reflect the unique protein environment and conformation of
the bound bilin. The observed spectral features (i.e. long
to short wavelength absorption ratio <1) indicate that bilins bind to
PcyA in a cyclic, porphyrin-like configuration, as opposed to the more
extended configurations found in phytochromes and phycobiliproteins
(17). This cyclic configuration precludes simultaneous protonation of
both B- and C-ring nitrogen atoms of the bilin prosthetic group due to
steric crowding (see Fig. 6). This
conclusion is further supported by the observed lack of fluorescence of
the PcyA-BV complex as efficient proton transfer between
hydrogen-bonded pyrrole rings would be expected to quench the excited
state of the PcyA-BV complex.
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Interestingly, a long wavelength shoulder was detected in the spectra
of the PcyA complexes of BV IX and BV XIII
. Since this shoulder
was not observed for BV III
, a nonmetabolized PcyA substrate analog,
we speculate that this new absorption band corresponds to a distinct
bilin-PcyA interaction, which reflects the ability of bilin to be
reduced (i.e. hydrogen bonding, protonation, or aromatic
-
interaction).
PcyA Prefers Plant Type [2Fe-2S] Ferredoxins--
Pioneering
work by Beale and Cornejo (18) has established that reduction of BV in
the rhodophyte C. caldarium is Fd-mediated. This result was
later confirmed with the cloning of the bilin reductase family and the
demonstration that all bilin reductases are Fd-dependent
enzymes (4, 10). The present studies revealed that PcyA exhibits a
preference for plant-type [2Fe-2S] Fds. Fldx, a two-electron acceptor
that also can undergo two successive low potential single electron
reductions (i.e. 413 mV at pH 7, 25 °C) (19), also
supported PcyA activity. Under the conditions tested here, the
Fldx-dependent activity was ~13% of that of Fd. More
recently, we have been able to increase the Fldx-mediated PcyA activity
up to 50% of that of Fd by increasing the pH of the assay buffer to
8.5 and adding 100 mM
KCl.3 Based on these results,
it is conceivable that Fldx can functionally substitute for Fd to drive
PcyA activity under iron-limiting conditions in vivo
(20).
The observation that the 2[4Fe-4S] ferredoxin from C. pasteurianum poorly supports PcyA-mediated BV reduction needs to
be interpreted with the following caveats. The PcyA assay requires a
spinach FNR-containing ferredoxin reducing system, which may be unable
to effectively reduce the C. pasteurianum Fd, thereby limiting PcyA activity. Since spinach Fd and C. pasteurianum
Fd have the same redox potential around 420 mV, C. pasteurianum Fd conceivably could support PcyA activity, assuming
the right reducing system is present. In this regard, C. pasteurianum Fd serves as an electron acceptor in the anaerobic
oxidation of pyruvate (21), the components of which may be able to
drive C. pasteurianum Fd-dependent PcyA activity.
The Endogenous Reductant for PcyA in E. coli May Be Flavodoxin-- Recent studies reporting the assembly of holophytochrome and holophycobiliproteins in E. coli (22-25) indicate that PcyA can use naturally occurring reductants in living cells. E. coli cells possess several possible reductants. E. coli Fd is an adrenodoxin-type [2Fe-2S] ferredoxin, which genetic analyses have shown performs an essential role in the maturation of various iron-sulfur proteins (26). Indeed, E. coli Fd is more structurally related to the adrenodoxin-type ferredoxins (i.e. bovine adrenodoxin and P. putida putidaredoxin) than to plant-type Fds (27). As such, E. coli Fd probably functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. Based on the observation that the PcyA-mediated catalysis is poorly supported by the putidaredoxin system (see Table I), we hypothesize that engineered PCB biosynthesis in E. coli uses a different reducing system. Other than this adrenodoxin-type ferredoxin, the E. coli genome possesses two Fldx genes and a flavorubredoxin gene (28). In light of the data presented here, we propose that the biosynthesis of PCB in E. coli is driven by one of the two Fldxs.
PcyA-mediated 18-Vinyl Reduction Precedes A-ring
Reduction--
The identification of
181,182-DHBV IX as an intermediate in the
conversion of BV to PCB has established that D-ring exovinyl reduction
precedes A-ring reduction. PcyA is therefore composed of two separate
activities mediated by a
181,182-DHBV:ferredoxin oxidoreductase and a
PCB:ferredoxin oxidoreductase. This double bond specificity of PcyA
presumably ensures that P
Bs are never produced in PcyA-containing
cyanobacteria or red algae, the production of which might lead to
misincorporation of P
Bs into their phycobiliproteins. We
speculate that P
B-containing phycobiliproteins would be more
susceptible to photooxidative damage than the natural PCB-containing
antennae of these organisms due to the presence of the reactive
exovinyl group on the former. Evolution of P
B-producing bilin
reductases, such as HY2, would therefore prove a selective disadvantage
to these organisms, a selection pressure that would not apply to
terrestrial plants that lack phycobiliproteins. In this regard, it will
be of interest to clone the genes for these enzymes from the green alga
M. caldariorum, which mediate the conversion of BV to PCB
via the intermediacy of P
B (5).
Through examination of substrate analogs, which include the unnatural
XIII and III
isomers of BV and the A-ring reduced phytochromobilin isomers, (3Z)- and (3E)-P
B,
our studies have provided insight into the catalytic specificity of
PcyA. Of the two unnatural BV analogs, only BV XIII
was metabolized
by PcyA, yielding the two-electron reduced iso-P
B product (both
(3Z)- and (3E)-isomers). Since BV XIII
is
symmetrical and lacks the exovinyl group found on BV IX
, this result
indicated that PcyA-mediated A-ring reduction can occur in the absence
of exovinyl reduction. The apparent lack of the reduction of the second
endovinyl group of BV XIII
also indicated that iso-P
B is a poor
PcyA substrate. By contrast to the IX
and XIII
isomers, BV III
was not metabolized by PcyA. This result was unexpected, given the
sequence of PcyA-mediated vinyl reductions of BV IX
. In this regard,
BV III
is symmetrical, possessing two exovinyl groups, one or both
of which should have been reduced by PcyA. Moreover, our results
indicate that BV III
binds to PcyA with the highest affinity of the
three BV isomers tested. These data indicate that bound BV III
is
not properly oriented within the enzyme's bilin binding site for
catalysis. Our studies show that (3Z)-P
B can be
metabolized by PcyA, yielding a mixture of PCB isomer products, whereas
(3E)-P
B is not a substrate for PcyA. These data indicate
a strong influence of the geometry of the 3-ethylidene moiety on
catalysis. Whether this is due to a positioning defect or to a lack of
binding of (3E)-P
B to PcyA remains to be determined.
Taken together, these studies suggest that proper substrate
positioning/activation within the enzyme is a prerequisite for catalysis.
A Radical Mechanism for Bilin Reduction by PcyA-- Four major Fd-dependent enzymes have been characterized to date: FNR, Fd:nitrite oxidoreductase, glutamate synthase, and Fd:thioredoxin reductase (29). All of these enzymes contain redox-active cofactors including FAD (FNR), iron-sulfur clusters (glutamate synthase, nitrite reductase, and sulfite reductase), and siroheme (nitrite reductase). By contrast with these Fd-dependent enzymes, PcyA appears to lack a metal or flavin cofactor that can mediate single electron transfers. For these reasons, we propose that the PcyA-mediated reduction of BV proceeds via bilin radical intermediates as depicted in Fig. 6.
Based upon the absorption spectrum of the PcyA·BV complex, the bilin substrate is depicted in a cyclic conformation within the protein cavity (see Fig. 6). The lack of photochromism of the PcyA·BV complex can be rationalized by the binding of the terminal pyrrolinone A- and D-rings into the protein matrix with its propionate side chains extending toward the solvent. This porphyrin-like configuration not only would sterically prevent photoisomerization of the C5 and C15 double bonds but would also bury reactive radical intermediates within the protein matrix, thus minimizing side reactions with molecular oxygen. This substrate binding model is consistent with the broad substrate specificity of the extended bilin reductase family, which includes the enzyme RCCR that metabolizes a chlorophyll catabolite with monomethyl ester and isocyclic ring substituents (30). In this regard, the hypothesis that the bilin reductase family may have evolved from ancestors that metabolized (Mg)-porphyrins remains a intriguing possibility.
As shown in Fig. 6 (step 1), we envisage that
bilin reduction occurs by binding of reduced Fd to the PcyA-BV complex,
followed by electron transfer to the bound bilin and proton transfer
from a protein residue labeled D1-H to generate a neutral
radical shown in step 2. The benzylic position
would help to stabilize this radical by resonance within the extended
tetrapyrrole -system, until a second electron and proton transfer,
shown in steps 2 and 3, occurs to
produce the intermediate 181,182-DHBV IX
.
The hypothetical proton donors, D1-H and D2-H,
could either be carboxylic acids (i.e. Asp or Glu),
sulfhydryls (i.e. Cys), phenolics (i.e. Tyr), or
even protonated nitrogen residues such as histidine or lysine. It is
also possible that protons are derived from bound water molecules that
are protonated by appropriate protein residues. For all of these
protein residues except for histidine or lysine, proton transfer would
be accompanied by an increase in negative charge, which would be a
reasonable "driving force" for the release of product. Since PcyA
kinetically reduces the intermediate
181,182-DHBV IX
without its release, we
hypothesize that D1 and D2 are protonated histidine and/or lysine residues.
We propose that the subsequent reduction of the A-ring of
181,182-DHBV IX proceeds in a similar
fashion, generating another resonance-stabilized bilin radical
intermediate shown in Fig. 6 (steps 4-6). For
this transformation, we hypothesize that the proton-donating residues are carboxylic acids, sulfhydryls, and/or phenolics, which would generate negative charge within the bilin pocket, thereby promoting release of the PCB product (Fig. 6, step 6).
Experiments to detect potential radical intermediates by electron spin
resonance spectroscopy and to identify putative proton-donating
residues within the PcyA polypeptide by site-directed mutagenesis are
in progress. With this experimental approach, we hope to elucidate the
molecular basis for the unique double bond reduction specificities of
the different members of the extended bilin reductase family (4) and
ultimately to engineer novel specificity of this important family of enzymes.
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ACKNOWLEDGEMENTS |
---|
We are grateful to James Partridge for
synthesis of 181,182-DHBV. We thank Drs.
D. A. Bryant and P. Ortiz de Montellano for providing clones
and Dr. D. A. Lightner for the gift of mBRIII. We thank Dr.
Shih-Long Tu and Amanda Ellsmore for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by United States Department of Agriculture Competitive Research Grant Initiative Grant AMD-0103397 (to J. C. L.) and by a fellowship from the Deutsche Forschungsgemeinschaft (to N. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 530-752-1865; Fax: 530-752-3085; E-mail: jclagarias@ucdavis.edu.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M211643200
2 C. Nishida and P. Ortiz de Montellano, personal communication.
3 S.-L. Tu, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
PCB, phycocyanobilin;
PB, phytochromobilin;
BR, bilirubin;
BV, biliverdin;
Cph1, cyanobacterial phytochrome 1;
DHBV, dihydrobiliverdin;
Fd, ferredoxin;
Fldx, flavodoxin;
FNR, ferredoxin-NADP+-oxidoreductase;
PcyA, phycocyanobilin:ferredoxin oxidoreductase;
HPLC, high pressure liquid
chromatography;
TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid.
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