The oxygen-independent coproporphyrinogen III oxidase HemN utilizes harderoporphyrinogen as a reaction intermediate during conversion of coproporphyrinogen III to protoporphyrinogen IX

Article abstract of DOI:10.1515/BC.2010.006

During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18- tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B. by Walter de Gruyter.

Full text of DOI:10.1515/BC.2010.006

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Biol. Chem., Vol. 391, pp. 55–63, January 2010 • Copyright ꢀ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2010.006
The oxygen-independent coproporphyrinogen III oxidase
HemN utilizes harderoporphyrinogen as a reaction
intermediate during conversion of coproporphyrinogen III
to protoporphyrinogen IX
Katrin Rand1, Claudia Noll2, Hans Martin Schiebel3,
Dorit Kemken4, Thomas Du¨lcks4, Markus Kalesse2,
Dirk W. Heinz1 and Gunhild Layer5,*
Introduction
Biosynthesis of hemes and (bacterio)chlorophylls requires
the conversion of coproporphyrinogen III to protoporphyri-
nogen IX. During this reaction the propionate side chains on
pyrrole rings A and B of coproporphyrinogen III are oxi-
datively decarboxylated to the corresponding vinyl groups of
protoporphyrinogen IX. Two molecules of CO2 are released
during the reaction and a final electron acceptor is required
to take up two electrons from each side chain (Figure 1). The
overall reaction is catalyzed by coproporphyrinogen III oxi-
dases (CPOs). There are two completely unrelated types of
CPOs in nature. In eukaryotes and some bacteria, oxidative
decarboxylation of coproporphyrinogen III is performed by
the oxygen-dependent CPO HemF. In most bacteria, how-
ever, the reaction is catalyzed by the oxygen-independent
enzyme HemN (Heinemann et al., 2008).
1 Division of Structural Biology, Helmholtz Center for
Infection Research, Inhoffenstrasse 7, D-38124
Braunschweig, Germany
2 Institute of Organic Chemistry, Leibniz University
Hannover, Schneiderberg 1b, D-30167 Hannover, Germany
3 Institute of Organic Chemistry, Technical University
Braunschweig, Hagenring 30, D-38106 Braunschweig,
Germany
4 Institute of Organic Chemistry, University of Bremen,
Leobener Strasse, NW2, D-28359 Bremen, Germany
5 Institute of Microbiology, Technical University
Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig,
Germany
*Corresponding author
e-mail: g.layer@tu-bs.de
HemF uses molecular oxygen as a final electron acceptor
(Breckau et al., 2003). Early studies of the oxidative decar-
boxylation of coproporphyrinogen III using radioactively
labeled substrates revealed that in the oxygen-dependent
reaction the propionate group on ring A is decarboxylated
prior to that on ring B, resulting in the intermediate harde-
roporphyrinogen (3-vinyl-8,13,17-tripropionic acid-2,7,12,
18-tetramethylporphyrinogen) (Sano and Granick, 1961;
Cavaleiro et al., 1973, 1974; Games et al., 1976; Elder et al.,
1978). Accordingly, exchanges of certain amino acid residues
probably involved in the second decarboxylation reaction
during HemF catalysis lead to accumulation of harderopor-
phyrin, the oxidized form of the reaction intermediate har-
deroporphyrinogen (Schmitt et al., 2005). This was first
observed in patients suffering from a rare variant form of
hereditary coproporphyria, so-called harderoporphyria
(Nordmann et al., 1983).
Abstract
During heme biosynthesis the oxygen-independent copro-
porphyrinogen III oxidase HemN catalyzes the oxidative
decarboxylation of the two propionate side chains on rings
A and B of coproporphyrinogen III to the corresponding
vinyl groups to yield protoporphyrinogen IX. Here, the
sequence of the two decarboxylation steps during HemN
catalysis was investigated. A reaction intermediate of HemN
activity was isolated by HPLC analysis and identified as
monovinyltripropionic acid porphyrin by mass spectrometry.
This monovinylic reaction intermediate exhibited identical
chromatographic behavior during HPLC analysis as harde-
roporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-
tetramethylporphyrin). Furthermore, HemN was able to
utilize chemically synthesized harderoporphyrinogen as sub-
strate and converted it to protoporphyrinogen IX. These
results suggest that during HemN catalysis the propionate
side chain of ring A of coproporphyrinogen III is decar-
boxylated prior to that of ring B.
The oxygen-independent CPO HemN belongs to the fam-
ily of radical SAM enzymes (Sofia et al., 2001). It catalyzes
the oxidative decarboxylation of coproporphyrinogen III via
a catalytic mechanism completely different to that of HemF.
HemF and HemN are also structurally completely unrelated.
HemN contains a catalytically essential w4Fe-4Sx cluster that
transfers an electron to bound S-adenosyl-L-methionine
(SAM), thereby producing methionine and a 59-desoxyade-
nosyl radical (Layer et al., 2002, 2005). This radical then
abstracts a hydrogen atom from the b-carbon of the substrate
propionate side chain, resulting in the formation of a copro-
porphyrinogenyl radical (Layer et al., 2006). Finally, decar-
boxylation and transfer of the remaining single electron to
Keywords: Escherichia coli; heme biosynthesis; oxidative
decarboxylation.
2010/272
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56 K. Rand et al.
Figure 1 Conversion of coproporphyrinogen III to protoporphyrinogen IX catalyzed by coproporphyrinogen III oxidases (CPOs).
CPOs catalyze the oxidative decarboxylation of the propionate side chains on rings A and B of coproporphyrinogen III to the corresponding
vinyl side chains of protoporphyrinogen IX. The oxygen-dependent CPO HemF catalyzes this reaction via the monovinylic intermediate
harderoporphyrinogen. For the oxygen-independent CPO HemN, the sequence of the two decarboxylation reactions has not been studied so
far.
an as yet unidentified final electron acceptor yields the vinyl
group of the reaction product. The crystal structure of Esche-
richia coli HemN has been solved, albeit without a bound
substrate or product (Layer et al., 2003). Although the oxi-
dized form of the substrate coproporphyrinogen III could be
placed manually into the active site pocket of HemN, it was
not possible to exclude one or the other orientation of the
substrate to predict the sequence of the two decarboxylation
reactions. So far, there are no biochemical reports on the
sequence of the decarboxylation reactions during oxygen-
independent protoporphyrinogen IX formation. Considering
that HemF and HemN are two structurally and catalytically
unrelated enzymes, both possible reaction intermediates,
harderoporphyrinogen and isoharderoporphyrinogen, are
conceivable for the HemN-catalyzed reaction.
the HemN-catalyzed reaction. Furthermore, we show that
HemN can utilize chemically synthesized harderoporphyri-
nogen as a substrate and converts it to protoporphyrinogen
IX. These results suggest that HemN, analogously to HemF,
catalyzes the conversion of coproporphyrinogen III to
protoporphyrinogen IX via the reaction intermediate
harderoporphyrinogen.
Results and discussion
A monovinyl-tripropionic acid porphyrinogen
is formed during HemN catalysis
To investigate the sequence of the two decarboxylation reac-
tions catalyzed by HemN, we performed HemN activity
assays and analyzed the porphyrin content in the reaction
mixtures after 15, 30, 45, 60 and 90 min of incubation by
HPLC. Porphyrins were detected fluorimetrically with exci-
In this study we address the question of the sequence of
the decarboxylation reactions during oxygen-independent
decarboxylation of coproporphyrinogen III. We show that a
monovinyl-tripropionic acid porphyrin is detectable during
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HemN reaction intermediate 57
Figure 2 (A) HPLC analysis of HemN and HemF R401K activity assays, (B) time-dependent formation of HemN reaction products, and
(C) ESI-MS analysis of porphyrins produced in the HemN activity assay.
(A) HemN and HemF R401K activity assays were performed as described in the text. Reactions were stopped after 30 min of incubation
and porphyrinogens were oxidized to the corresponding porphyrins for HPLC analysis. Chromatogram (a) shows the standard HemN activity
assay, chromatogram (b) the same activity assay without HemN and chromatogram (c) the HemF R401K activity assay. Retention times for
the different porphyrins were 6.6 min for coproporphyrin III, 39 min for protoporphyrin IX and 10.8 min for harderoporphyrin. Copropor-
phyrin III (Frontier Scientific Europe) and protoporphyrin IX (Sigma) were used as HPLC standards. Porphyrins were detected by fluores-
cence (excitation at 409 nm, emission at 630 nm). (B) Porphyrins present in HemN activity assays after 15, 30, 45, 60 and 90 min of
incubation were analyzed by HPLC. The area wmV=sx for peaks corresponding to coproporphyrin III (j), protoporphyrin IX (m) and the
monovinyl-tripropionic acid porphyrin (d) was plotted against the reaction time. (C) Porphyrins present in a HemN activity assay after
60 min of incubation were analyzed by electrospray mass spectrometry (ESI-MS). Mass spectra were recorded in negative ion mode. ESI-
-
MS analysis revealed the presence of coproporphyrin III with a mass of 653.3 for the wM-Hx ion, of protoporphyrin IX with a mass of
561.5 and of a monovinyl-tripropionic acid porphyrin with a mass of 607.5.
tation at 409 nm and emission at 630 nm. Three major peaks
were observed in HPLC chromatograms for the standard
HemN activity assay (Figure 2A). The oxidized HemN sub-
strate coproporphyrin III eluted at a retention time of
6.6 min, whereas the oxidized HemN product protoporphyrin
IX was detected at a retention time of approximately 39 min.
In addition to the peaks for these expected porphyrins, a third
peak corresponding to a fluorescent molecule was observed
at a retention time of 10.8 min. This molecule was identified
as porphyrin by photometric diode array analysis. In a con-
trol reaction in which HemN was omitted from the assay
mixture, only minor formation of protoporphyrin IX and the
additional porphyrin was observed. This residual activity was
presumably due to the presence of CPOs in the E. coli cell-
free extract necessary for the electron acceptor activity in
our assay. In the HemN-containing activity assay, the
amounts of the new porphyrin remained almost constant after
reaction times of 15, 30, 45 and 60 min, whereas the copro-
porphyrinogen III concentration decreased and the protopor-
phyrinogen IX concentration increased (Figure 2B). Since
the retention time of this new porphyrin was between those
of coproporphyrin III and protoporphyrin IX, we suspected
that it was a monovinyl-tripropionic acid porphyrin. This is
the oxidized form of a potential HemN reaction intermediate,
in which one of the propionate side chains on ring A or B
is already decarboxylated to the corresponding vinyl group.
To verify this hypothesis, the porphyrins present in a HemN
activity assay after 60 min of incubation were extracted from
the reaction mixture and subjected to electrospray mass spec-
trometry (ESI-MS). Mass spectra were recorded in negative
ion mode (Figure 2C). As expected, ESI-MS analysis
revealed the presence of coproporphyrin III with a mass of
-
653.3 for the wM-Hx ion and of protoporphyrin IX with a
mass of 561.5. In addition, a mass of 607.5 was observed,
which corresponds to the mass of a monovinyl-tripropionic
acid porphyrin.
These results demonstrate that the two propionate side
chains on rings A and B of coproporphyrinogen III are
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58 K. Rand et al.
sequentially decarboxylated during the HemN-catalyzed
reaction, as is the case for the oxygen-dependent CPO HemF,
and not simultaneously. However, from the experiments
described above it was not clear whether the ring A or ring
B propionate side chain was decarboxylated first.
wig and Lehr, 2004). A slightly modified protocol was used
for condensation of pyrromethanes 3 and 5 to avoid large
amounts of self-condensation product 8 of the southern frag-
ment 5. In this step the equivalents of the coupling reagent
p-toluenesulfonic acid were changed from 7.5 to 5 eq. and
the time over which dialdehyde 5 was added to dipyrrole 3
was changed to 14 h. To investigate whether HemN can
accept harderoporphyrinogen as a substrate and convert it to
protoporphyrinogen IX, we hydrolyzed harderoporphyrin tri-
methylester with HCl and reduced the resulting porphyrin
using sodium amalgam. The harderoporphyrinogen solution
obtained was analyzed by HPLC. Three peaks were observed
in the HPLC chromatogram (Figure 4). The major peak cor-
responds to harderoporphyrin, with a retention time of
10.8 min. The two additional peaks probably correspond to
by-products of hydrolysis and sodium amalgam reduction, at
retention times of 9 and 6.5 min. These contaminants were
not further investigated; however, it has been reported that
by-products are formed during hydrolysis of porphyrins con-
taining a vinyl group in HCl solution and during sodium
amalgam reduction (Falk et al., 1956). Therefore, the sub-
strate solution used for the HemN activity assays consisted
of a mixture of harderoporphyrinogen and two porphyrino-
gens that were not further characterized (Figure 4).
HemN activity assays with harderoporphyrinogen as the
substrate were performed as described in the materials and
methods section. After oxidation of the reaction products, the
porphyrin content of the assay mixtures was analyzed by
HPLC (Figure 4). In the assay containing HemN, we
observed the formation of protoporphyrinogen IX with a
concomitant decrease in harderoporphyrinogen. In a control
reaction in which HemN was omitted from the mixture, only
minor protoporphyrinogen IX formation from harderopor-
phyrinogen was observed. This residual activity originated
from CPOs present in the E. coli cell-free extract used for
the activity assay and was also observed in activity assays
with coproporphyrinogen III as substrate (see above). In
another control reaction in which no HemN and no E. coli
cell-free extract were present, we did not observe any pro-
toporphyrinogen IX formation. From the HPLC chromato-
grams in Figure 4 it is evident that HemN can accept
harderoporphyrinogen as a substrate and convert it to pro-
toporphyrinogen IX. By contrast, the two contaminating por-
phyrinogens present in the substrate solution (see above)
were not significantly further metabolized by HemN.
The monovinyl-tripropionic acid porphyrin produced
by HemN shows the same chromatographic behavior
as harderoporphyrin produced by HemF
For the oxygen-dependent CPO HemF it is well established
that the ring A propionate side chain is decarboxylated first,
resulting in formation of the reaction intermediate hardero-
porphyrinogen (Sano and Granick, 1961; Cavaleiro et al.,
1974, 1973; Games et al., 1976; Elder et al., 1978). More-
over, several HemF mutant proteins have been described for
which a single amino acid exchange leads to accumulation
of harderoporphyrinogen during activity assays. For exam-
ple, the human HemF variant R401K is a harderoporphyri-
nogen-accumulating protein (Schmitt et al., 2005). To obtain
a first clue as to whether the monovinyl-tripropionic acid
porphyrin produced in HemN activity assays is harderopor-
phyrin, we compared the chromatographic behavior of the
porphyrin produced by HemN and harderoporphyrin pro-
duced by HemF R401K using reverse-phase HPLC. We pro-
duced the recombinant human HemF mutant protein R401K
in E. coli and purified the protein as a GST fusion protein.
We performed activity assays with HemF R401K, analyzed
the porphyrin content of the enzyme reactions by HPLC and
compared the chromatograms obtained for HemF R401K and
HemN activity assays. As expected, three major peaks in
HPLC chromatograms of HemF R401K activity assays rep-
resented coproporphyrin III, protoporphyrin IX and harde-
roporphyrin (Figure 2A). More importantly, the retention
time of harderoporphyrin produced in the HemF R401K
activity assay was identical to that of the monovinyl-tripro-
pionic acid porphyrin produced in the HemN activity assay.
This result strongly suggests that the monovinylic HemN
reaction intermediate observed is indeed harderoporphyri-
nogen. However, in the absence of an isoharderoporphyrin
HPLC standard we cannot exclude the possibility that both
harderoporphyrin and isoharderoporphyrin exhibit identical
chromatographic behavior during the HPLC assay we used.
Therefore, to substantiate our assumption that HemN
catalyzes the decarboxylation of coproporphyrinogen III to
protoporphyrinogen IX via the reaction intermediate harde-
roporphyrinogen, we investigated whether HemN can use
chemically synthesized harderoporphyrinogen as a substrate
and convert it to protoporphyrinogen IX.
These results clearly demonstrate that HemN can convert
harderoporphyrinogen to protoporphyrinogen IX. The obser-
vations described above strongly suggest that HemN cata-
lyzes the decarboxylation of the ring A propionate side chain
of coproporphyrinogen III prior to the second decarboxyla-
tion on ring B with harderoporphyrinogen as a reaction inter-
mediate, although we cannot completely exclude the
possibility that HemN could also accept isoharderoporphy-
rinogen as a substrate. Co-crystallization of HemN with
coproporphyrinogen III would provide the necessary insight
into the orientation of the substrate and its propionate side
chains in the active site of the enzyme. Considering our find-
ings that harderoporphyrinogen is a possible HemN reaction
HemN can convert chemically synthesized
harderoporphyrinogen to protoporphyrinogen IX
Harderoporphyrin trimethyl ester 7 was synthesized by con-
densation of pyrromethanes 3 and 5 following a modified
MacDonald approach that constructs the tetrapyrrole in a
convergent approach from a northern and southern segment
(Figure 3) (Arsenault et al., 1960; Carr et al., 1971; Cavaleiro
et al., 1973, 1974; Chen et al., 1999; Lash et al., 1999; Lud-
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HemN reaction intermediate 59
Figure 3 Synthesis of harderoporphyrin trimethylester.
Harderoporphyrin trimethyl ester (7) was synthesized by condensation of pyrromethanes (3) and (5) following the modified MacDonald
approach with some minor modifications as described in the text (Arsenault et al., 1960; Carr et al., 1971; Cavaleiro et al., 1973, 1974;
Chen et al., 1999; Lash et al., 1999; Ludwig and Lehr, 2004).
ammonium acetate were purchased from Fisher Scientific GmbH
(Schwerte, Germany). Coproporphyrin III was purchased from Fron-
tier Scientific Europe (Carnforth, UK).
intermediate, the ring A propionate side chain of copropor-
phyrinogen III is expected to be found in close proximity to
the w4Fe-4Sx-bound S-adenosyl-L-methionine. Further bio-
chemical and crystallographic studies are needed to under-
stand how the reaction intermediate harderoporphyrinogen is
further decarboxylated.
Plasmids, bacteria and growth conditions
Plasmids for production of recombinant E. coli HemN and recom-
binant human HemF variant R401K were pET3ahemN (Layer et al.,
2002) and pGEX-2T:CPO(R401K) (Schmitt et al., 2005), respec-
tively. E. coli BL21(DE3) or E. coli BL21(DE3) Codon Plus was
used as the host for protein production. Growth conditions for
recombinant HemN production were as previously described (Layer
et al., 2002). For production of recombinant human HemF variant
R401K, E. coli BL21(DE3) Codon Plus cells carrying plasmid
pGEX-2T:CPO(R401K) were grown in LB medium at 378C. Gene
Materials and methods
Chemicals
Unless otherwise stated, chemicals, reagents and antibiotics were
obtained from Sigma-Aldrich (Taufkirchen, Germany) or Merck
(Darmstadt, Germany). HPLC-grade methanol, acetonitrile and
expression was induced with 50 m
M isopropyl-b-D-thiogalactopy-
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60 K. Rand et al.
HemN activity assay
The HemN activity assay was performed under anaerobic conditions
in an anaerobic chamber (Coy Laboratories, Grass Lake, MI, USA)
as previously described (Layer et al., 2002). The standard assay
mixtures contained 100 m
100, 500 m NADH, 500 m
extract (prepared as described by Layer et al., 2002), 3 m
300 m NaCl and 20 m coproporphyrinogen III in a total volume
of 100 ml. When coproporphyrinogen III was used as the substrate
the final HemN concentration was 1.5 m . When chemically syn-
thesized harderoporphyrinogen was used as the substrate the final
HemN concentration was 15 m . The assay mixtures were incu-
M
Bis-Tris, pH 7.0, 0.3% (v/v) Triton X-
SAM, 15 ml of E. coli cell-free
DTT,
M
M
M
M
M
M
M
bated at 378C. After defined points of time the reactions were
stopped by the addition of 5 ml H2O2 (30%). After 15 min of incu-
bation the samples were flash frozen in liquid nitrogen and stored
at -208C until further use.
HemF activity assay
The HemF activity assay was performed in a total volume of 100 ml
of 100 m
3 m DTT, 300 m
final HemF concentration was 5 m
M
Bis-Tris, pH 7.0 containing 0.3% (v/v) Triton X-100,
NaCl and 20 m coproporphyrinogen III. The
. After 30 or 60 min of incu-
M
M
M
Figure 4 HPLC analysis of HemN activity assays with hardero-
porphyrinogen as the substrate.
M
bation at 378C the reaction was stopped by addition of 5 ml of H2O2
(30%). After 15 min of incubation the samples were flash-frozen in
liquid nitrogen and stored at -208C until further use.
Activity assays with harderoporphyrinogen as the substrate were
performed as described in the text. Reactions were stopped after
60 min of incubation and porphyrinogens were oxidized to the cor-
responding porphyrins. (A) HPLC analysis of porphyrins in a con-
trol reaction in which no HemN and no E. coli cell-free extract were
present. (B) Analysis of porphyrins in a control reaction with an E.
coli cell-free extract in the absence of HemN. Only minor proto-
porphyrinogen IX formation from harderoporphyrinogen was
observed. (C) HPLC analysis of porphyrins in a sample containing
HemN and E. coli cell-free extract. Formation of protoporphyrin IX
(39 min) was observed with a concomitant decrease in harderopor-
phyrin (10.8 min).
Coproporphyrinogen III preparation
Coproporphyrinogen III was prepared by chemical reduction of
coproporphyrin III with 3% sodium amalgam as previously
described (Layer et al., 2002).
Harderoporphyrinogen preparation
Harderoporphyrinogen was generated by reduction of harderopor-
phyrin with 3% sodium amalgam. In brief, harderoporphyrin was
ranoside at an OD600 of 0.6 and the cells were further cultivated at
258C overnight to allow recombinant protein production.
dissolved in 250 ml of 10 m
M KOH and heated to 808C. Freshly
prepared 3% sodium amalgam (0.6 g) was added to the hot harde-
roporphyrin solution and the mixture was stirred for 5 min in the
dark. The almost colorless solution was filtered through glass wool
Enzyme purification
and 550 ml of buffer (1
The solution was adjusted to pH 8.0 by adding several drops of 5
HCl. The harderoporphyrinogen solution was stored at -208C until
further use.
M Tris-HCl, pH 7.5, 5 mM DTT) was added.
Recombinant E. coli HemN was purified as previously described
(Layer et al., 2002). Recombinant human HemF variant R401K was
purified as a glutathione S-transferase (GST) fusion protein on glu-
tathione-Sepharose (GE Healthcare, Mu¨nchen, Germany) according
to the manufacturer’s instructions. In brief, after protein production
E. coli cells were harvested by centrifugation, the cell pellet was
M
Harderoporphyrin preparation
resuspended in buffer A w50 m
M Tris, pH 8.0, 100 mM NaCl, 3 mM
Harderoporphyrin was obtained from harderoporphyrin trimethyl
dithiothreitol (DTT)x and cells were disrupted by sonication. The
soluble protein fraction was incubated with glutathione-Sepharose
for 1 h at 48C. The affinity material was then placed in a small
column and non-bound proteins were washed off the column with
buffer A. The fusion protein GST-HemF R401K was eluted using
ester by hydrolysis with 5
M HCl for 6 h at room temperature.
Following hydrolysis, the pH of the porphyrin solution was adjusted
to 4 by adding saturated sodium acetate. The precipitated porphyrin
was sedimented by centrifugation and washed with water. The por-
phyrin sediment was dissolved in 5 ml of diethyl ether and harde-
buffer A supplemented with 10 m
solutions were concentrated by ultrafiltration (Amicon, Millipore
GmbH, Eschborn, Germany) and stored at -208C.
M reduced glutathione. Protein
roporphyrin was extracted from the ether with 0.15
M HCl. The
harderoporphyrin solution obtained was freeze-dried and the por-
phyrin was stored at -208C until further use.
Protein concentration
Synthesis of harderoporphyrin trimethyl ester
Bradford reagent (Sigma-Aldrich) was used to determine protein
concentrations according to the manufacturer’s instructions using
bovine serum albumin as a standard.
Harderoporphyrin trimethyl ester (7) was synthesized by conden-
sation of pyrromethanes (3) and (5) (Figure 3) following the mod-
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HemN reaction intermediate 61
ified MacDonald approach (Arsenault et al., 1960; Cavaleiro et al.,
1974) with some minor modifications. Pyrromethanes (1) and (4)
were synthesized as previously described (Carr et al., 1971; Chen
et al., 1999; Lash et al., 1999; Ludwig and Lehr, 2004).
tert-Butyl-59-(carboxyacid)-39,4-bis(2-methoxycarbonylethyl)-3,49-di-
methyl-2,29-dipyrryl-methane-5-carboxylate (2): Dipyrryl (1)
(749 mg, 1.35 mmol) was dissolved in MeOH (80 ml) and 10%
palladium on charcoal (75 mg) was added. The reaction was stirred
at room temperature under a hydrogen atmosphere for 20 h. The
catalyst was removed by filtration through a short plug of Celite,
which was washed with MeOH. The solvent was removed under
reduced pressure to yield 2 as red crystals (599 mg, 1.28 mmol,
95%).
the reaction was set aside for 10 h. The mixture was washed with
brine (25 ml), NaHCO3 (aq.) (25 ml), and brine (25 ml), dried over
Na2SO4 and evaporated to dryness. The residue was treated with
5% H2SO4 in MeOH (20 ml) for 14 h in the dark and then diluted
with CH2Cl2 (40 ml). The organic layer was successively washed
with NaOAc(aq.) (15 ml), NaHCO3 (aq.) (15 ml), and brine (25 ml),
dried over Na2SO4, filtered and evaporated. The residue was puri-
fied by flash chromatography on silica gel (EtOAc/CH2Cl2 1:6) to
give the chloroethylporphyrin 6 (22.3 mg, 32.4 mmol, 33%) as red
crystals.
Melting point 173–1748C. Rfs0.55 (CH2Cl2/EtOAc 6:1). 1H
NMR (400 MHz, CDCl3): d 10.00 (s, 3H), 10.02 (s, 1H), 4.56–4.24
(m, 6H, 3=CH2), 3.64 (s, 9H, 3=OMe), 3.31–3.14 (m, 4H,
2=CH2), 2.67–2.49 (m, 2H, CH2), 2.43–2.18 (m, 4H, 2=CH2), 1.20
(m, 12H, 4=CH3), -3.79 (brs, 2NH). HR-ESI-MS: calcd. for
C38H44ClN4O6 wMqHxq 687.2849, found 687.2946.
Harderoporphyrin trimethyl ester (7): Chloroporphyrin (6)
(11.8 mg, 17.2 mmol) was dissolved in CH2Cl2 (4.9 ml) and treated
in the dark with a saturated methanolic Zn(OAc)2 solution (1.5 ml).
After 2 h, water (3 ml) was added and the zinc complex was extract-
ed with CH2Cl2. The organic layer was dried over Na2SO4 and fil-
tered and the solvent was removed under reduced pressure. The
residue was dissolved in tetrahydrofuran (2.1 ml) followed by addi-
Melting point 145–1488C. Rfs0.22 (CH2Cl2/EtOAc 5:2). 1H
NMR (200 MHz, CDCl3): d 11.54 (br s, 1H), 10.76 (br s, 1H), 3.87
(s, 2H), 3.66 (s, 3H), 3.38–3.27 (m, 2H), 3.02–2.81 (m, 4H),
2.55–2.40 (m, 2H), 2.29 (s, 3H), 2.11 (s, 3H), 1.53 (s, 9H). 13C
NMR (100 MHz, CDCl3): d 173.7, 166.1, 163.3, 133.5, 131.3,
129.7, 128.7, 119.2, 118.2, 118.0, 116.3, 82.4, 51.7, 43.9, 35.3, 28.6,
28.5, 22.8, 21.5, 11.0, 9.1. HR-EI-MS: calcd. for C22H31ClN2O4
wM-COOxqØ 422.1972, found 422.1975.
1H-Dipyrrole (3): Trifluoroacetic acid (TFA, 11 ml) was added
to 2 (264 mg, 566 mmol) at 08C and the solution was stirred for
3 h. The solvent was then evaporated under reduced pressure. The
residual oil was dissolved in CH2Cl2 (10 ml) and washed succes-
sively with H2O (5 ml), NaHCO3 (aq.) (5 ml) and H2O (5 ml). The
organic layer was dried over Na2SO4 and concentrated and the res-
idue was purified by flash chromatography on silica gel (100%
EtOAc) to provide 3 (123.6 mg, 382.9 mmol, 68%).
tion of 1
M KOtBu solution in tBuOH (4.5 ml) and stirred for 72 h
in the dark. To this solution pyridine (1.9 ml), EtOAc (0.9 ml) and
CHCl3 (10 ml) were added and the resulting mixture was then trans-
ferred to a separatory funnel with H2O (10 ml). The organic layer
was separated, extracted with EtOAc (15 ml), dried over Na2SO4
and evaporated. The dark brown residue was dissolved in 5% H2SO4
in MeOH (15.7 ml) and stirred in the dark for 14 h. The solution
was diluted with CH2Cl2 (12 ml) and washed with NaOAc(aq.)
(15 ml), NaHCO3(aq.) (15 ml) and finally brine (10 ml). The organic
layer was dried over Na2SO4, filtered and evaporated. The residue
was purified by flash chromatography on silica gel with CH2Cl2 as
the solvent (the column was covered with aluminum foil to limit
exposure to light). Compound 7 was obtained as deep red needles
(8.7 mg, 13.4 mmol, 78%).
1
Rfs0.60 (CH2Cl2/EtOAc 10:1). H NMR (400 MHz, CDCl3): d
8.74 (br s, 1H), 7.69 (br s, 1H), 6.52 (d, Js2.6 Hz, 1H), 6.48 (d,
Js2.6 Hz, 1H), 3.94 (s, 2H), 3.67 (s, 3H), 3.49 (t, Js7.1 Hz, 3H),
2.91–2.85 (m, 2H), 2.79–2.73 (m, 2H), 2.59–2.53 (m, 2H), 2.33 (s,
3H), 2.04 (s, 3H). 13C NMR (200 MHz, CDCl3): d 174.0, 126.5,
124.7, 122.0, 118.3, 116.0, 115.1, 114.3, 113.4, 51.6, 45.1, 34.9,
28.2, 23.2, 21.2, 10.4, 8.9. HR-EI-MS: calcd. for C17H23ClN2O2
wMxqØ 322.1448, found 322.1450.
Melting point 217–2198C. Rfs0.50 (CH2Cl2). 1H NMR
(400 MHz, CDCl3): d 10.23 (s, 1H), 10.16 (s, 1H), 10.09 (s, 1H),
10.07 (s, 1H), 8.29 (dd, Js17.8, 11.4 Hz, 1H), 6.34 (d, Js17.7 Hz,
1H), 6.17 (d, Js11.3 Hz, 1H), 4.47–4.36 (m, 4H, 2=CH2),
3.78–3.58 (m, 9H, 3=OMe), 3.31–3.22 (m, 4H, 2=CH2), 2.39–2.24
(m, 4H, 2=CH2), 1.50–1.40 (m, 12H, 4=Me), -3.68 (s, 2NH). HR-
ESI-MS: calcd. C38H43N4O6 wMqHxq 651.3183, found 651.3182.
5,59-Diformyl-4,49-dimethyl-3,39-bis-(methoxycarbonylethyl-2,29-
dipyrrylmethane (5): 5,59-bis(tert-butoxycarbonyl)-4,49-dimethyl-
3,39-bis-(methoxycarbonylethyl)-2,29-dipyrryl-methane (4) (2.37 g,
4.33 mmol) was dissolved in TFA (13 ml) and stirred at 08C for
30 min. Trimethyl orthoformate (4.13 g, 4.27 ml, 38.98 mmol) was
added and stirred for an additional 20 min. The mixture was
quenched with brine (60 ml) and diluted with ammonia (10%) to
adjust the pH to 8. The organic layer was separated and the aqueous
layer was extracted with EtOAc (5=40 ml). The combined organic
layers were dried over Na2SO4, filtered and evaporated under
reduced pressure. Purification using flash chromatography (100%
EtOAc) on silica gel provided aldehyde 5 (528 mg, 1.30 mmol,
30%) as brown crystals.
Melting point 179–1808C. Rfs0.41 (EtOAc). 1H NMR
(400 MHz, CDCl3) d 10.01 (br s, 2NH), 9.48 (s, 2H), 4.04 (s, 2H),
3.71 (s, 6H), 2.80 (t, Js7.1 Hz, 4H), 2.55 (t, Js7.1 Hz, 4H), 2.29
(s, 6H). 13C NMR (100 MHz, CDCl3): d 176.9, 174.0, 138.1, 135.2,
129.2, 121.2, 52.2, 34.3, 22.6, 19.1, 9.0. HR-EI-MS: calcd. for
C21H26N2O6 wMxqØ 402.1791, found 402.1788.
HPLC analysis
Reaction products of HemN and HemF activity assays were ana-
lyzed by HPLC. Activity assays were performed as described above.
For protein precipitation, 5 ml of HCl (37% w/v) was added to the
H2O2 oxidized reaction mixtures and precipitated proteins were
removed by centrifugation. The resulting supernatant (100 ml) was
mixed with 100 ml of acetone/HCl (97.5:2.5 v/v) and the sample
was centrifuged for 3 min. A 20-ml aliquot of the resulting super-
natant was loaded onto a 4.6=250 mm Equisil BDS C18-2 reverse-
phase column (Dr. Maisch GmbH, Ammerbuch, Germany; particle
size 5 mm). Porphyrin separation was performed at a flow rate of
2-Chloroethylporphyrin (6): 1H-Dipyrrole (3) (31.3 mg,
97.0 mmol) was dissolved in CH2Cl2 (22 ml) and stirred in the dark
with pTsOH (92.2 mg, 484.8 mmol). A solution of the dipyrryl (5)
(38.6 mg, 95.9 mmol) in CH2Cl2 (10 ml) was added dropwise to
the reaction mixture over 14 h. After complete addition the resulting
dark red solution was stirred at room temperature for 2 h. A satu-
rated solution of zinc acetate in methanol (1.5 ml) was added and
0.5 ml/min using 17% (v/v) ammonium acetate (1
M, pH 5.2) and
10% (v/v) acetonitrile in methanol as mobile phase. Porphyrins were
detected by fluorescence measurement using an excitation wave-
length of 409 nm and an emission wavelength of 630 nm and in
parallel by UV photometric diode array over the range 200–650 nm.
The HPLC system was a Jasco 1500 series (Jasco, Groß-Umstadt,
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62 K. Rand et al.
Germany). Coproporphyrin III and protoporphyrin IX were used as
porphyrin standards.
Cavaleiro, J.A., Kenner, G.W., and Smith, K.M. (1973). Biosyn-
thetic intermediates between coproporphyrinogen-III and proto-
porphyrinogen-IX. J. Chem. Soc. Chem. Commun. 5, 183–184.
Cavaleiro, J.A., Kenner, G.W., and Smith, K.M. (1974). Pyrroles
and related compounds. XXXII. Biosynthesis of protoporphyrin-
IX from coproporphyrinogen-III. J. Chem. Soc. Perkin 1 10,
1188–1194.
Chen, Q., Huggins, M.T., Lightner, D.A., Norona, W., and Mc-
Donagh, A.F. (1999). Synthesis of a 10-oxo-bilirubin: effects of
the oxo group on conformation, transhepatic transport, and glu-
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Elder, G.H., Evans, J.O., Jackson, J.R., and Jackson, A.H. (1978).
Factors determining the sequence of oxidative decarboxylation
of the 2- and 4-propionate substituents of coproporphyrinogen
III by coproporphyrinogen oxidase in rat liver. Biochem. J. 169,
215–223.
Porphyrin extraction for mass spectrometry
Porphyrins were extracted from activity assay mixtures as previ-
ously described using ethyl acetate/acetic acid (3:1 v/v) (Layer et
al., 2006). In the final step of this procedure the porphyrins in the
ethyl acetate/acetic acid fraction were extracted with 15% (w/v)
HCl. The pH of this HCl solution was adjusted to 4.0 by addition
of saturated sodium acetate solution to precipitate the porphyrins.
After centrifugation the precipitated porphyrins were washed once
with H2O. The remaining porphyrin sediment after centrifugation
was freeze-dried and stored at -208C until further use.
Mass spectrometry
Falk, J.E., Dresel, E.I., Benson, A., and Knight, B.C. (1956). Studies
on the biosynthesis of blood pigments. 4. The nature of the por-
phyrins formed on incubation of chicken erythrocyte prepara-
tions with glycine, d-aminolaevulic acid or porphobilinogen.
Biochem. J. 63, 87–94.
Games, D.E., Jackson, A.H., and Jackson, J.R. (1976). Biosynthesis
of protoporphyrin-IX from coproporphyrin-III. J. Chem. Soc.
Chem. Commun. 6, 187–188.
Heinemann, I.U., Jahn, M., and Jahn, D. (2008). The biochemistry
of heme biosynthesis. Arch. Biochem. Biophys. 474, 238–251.
Lash, T.D., Mani, U.N., Drinan, M.A., Zhen, C., Hall, T., and Jones,
M.A. (1999). Normal and abnormal heme biosynthesis. I.
Synthesis and metabolism of di- and monocarboxylic porphyri-
nogens related to coproporphyrinogen-III and harderoporphyri-
nogen: a model for the active site of coproporphyrinogen
oxidase. J. Org. Chem. 64, 464–477.
LR-ESI-MS data were acquired on a Bruker Esquire instrument
(Bruker Daltonik, Bremen, Germany). Samples were dissolved in
methanol and introduced by direct infusion at a flow rate of 3 ml/
min. HR-ESI-MS data were recorded on a Micromass LCT instru-
ment (Waters GmbH, Eschborn, Germany). HR-EI-MS measure-
ments were performed on a Finnigan MAT 711 instrument (Finni-
gan MAT, Bremen, Germany). Data were obtained by peak match-
ing at a resolution of approximately 10 000 (10% valley definition).
NMR spectroscopy
1H NMR spectra were recorded on a Bruker DPX-200 or DPX-400
spectrometer (Bruker Analytische Messtechnik, Rheinstetten, Ger-
many). 13C NMR spectra were recorded on a Bruker AVS-400
instrument (Bruker Analytische Messtechnik). Deuterochloroform
or tetramethylsilane was used as the internal standard.
Layer, G., Verfu¨rth, K., Mahlitz, E., and Jahn, D. (2002). Oxygen-
independent coproporphyrinogen-III oxidase HemN from Esche-
richia coli. J. Biol. Chem. 277, 34136–34142.
Layer, G., Moser, J., Heinz, D.W., Jahn, D., and Schubert, W.D.
(2003). Crystal structure of coproporphyrinogen III oxidase
reveals cofactor geometry of radical SAM enzymes. EMBO J.
22, 6214–6224.
Layer, G., Grage, K., Teschner, T., Schu¨nemann, V., Breckau, D.,
Masoumi, A., Jahn, M., Heathcote, P., Trautwein, A.X., and
Jahn, D. (2005). Radical S-adenosylmethionine enzyme copro-
porphyrinogen III oxidase HemN: functional features of the
Acknowledgments
We thank Prof. Jean-Charles Deybach and Caroline Schmitt (Uni-
versity of Paris VII, France) for the gift of plasmids pGEX-2T:CPO
and pGEX-2T:CPO(R401K). We would like to thank Prof. Dieter
Jahn for continuous support and helpful discussions. This work was
supported by grants from the Deutsche Forschungsgemeinschaft to
D.W.H. and by the Emmy-Noether-Program of the Deutsche For-
schungsgemeinschaft to G.L. Funds from the Fonds der Chemischen
Industrie are gratefully acknowledged by G.L. and D.W.H.
w4Fe-4Sx cluster and the two bound S-adenosyl-
J. Biol. Chem. 280, 29038–29046.
L-methionines.
Layer, G., Pierik, A.J., Trost, M., Rigby, S.E., Leech, H.K., Grage,
K., Breckau, D., Astner, I., Ja¨nsch, L., Heathcote, P., et al.
(2006). The substrate radical of Escherichia coli oxygen-inde-
pendent coproporphyrinogen III oxidase HemN. J. Biol. Chem.
281, 15727–15734.
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Received September 4, 2009; accepted September 24, 2009
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