Molecular hijacking of siroheme for the synthesis of heme and d1 heme.
ABSTRACT Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B(12), coenzyme F(430), and heme d(1) underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d(1) heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d(1) heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.
- SourceAvailable from: Susan M Lea[Show abstract] [Hide abstract]
ABSTRACT: It has recently been shown that the biosynthetic route for both the d1 -haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1 -haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.Molecular Microbiology 04/2014; 92(1):153-63. · 4.96 Impact Factor
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ABSTRACT: An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the D. vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5´-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe-4S](2+/1+) centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induce conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.Biochimica et Biophysica Acta 04/2014; · 4.66 Impact Factor
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ABSTRACT: Hemes (a, b, c, and o) and heme d 1 belong to the group of modified tetrapyrroles, which also includes chlorophylls, cobalamins, coenzyme F430, and siroheme. These compounds are found throughout all domains of life and are involved in a variety of essential biological processes ranging from photosynthesis to methanogenesis. The biosynthesis of heme b has been well studied in many organisms, but in sulfate-reducing bacteria and archaea, the pathway has remained a mystery, as many of the enzymes involved in these characterized steps are absent. The heme pathway in most organisms proceeds from the cyclic precursor of all modified tetrapyrroles uroporphyrinogen III, to coproporphyrinogen III, which is followed by oxidation of the ring and finally iron insertion. Sulfate-reducing bacteria and some archaea lack the genetic information necessary to convert uroporphyrinogen III to heme along the "classical" route and instead use an "alternative" pathway. Biosynthesis of the isobacteriochlorin heme d 1, a cofactor of the dissimilatory nitrite reductase cytochrome cd 1, has also been a subject of much research, although the biosynthetic pathway and its intermediates have evaded discovery for quite some time. This review focuses on the recent advances in the understanding of these two pathways and their surprisingly close relationship via the unlikely intermediate siroheme, which is also a cofactor of sulfite and nitrite reductases in many organisms. The evolutionary questions raised by this discovery will also be discussed along with the potential regulation required by organisms with overlapping tetrapyrrole biosynthesis pathways.Cellular and Molecular Life Sciences CMLS 02/2014; · 5.62 Impact Factor
Molecular hijacking of siroheme for the
synthesis of heme and d1heme
Shilpa Balia,b, Andrew D. Lawrenceb, Susana A. Lobob,c, Lígia M. Saraivac, Bernard T. Goldingd, David J. Palmerb,
Mark J. Howardb, Stuart J. Fergusona,1, and Martin J. Warrenb,1
aDepartment of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom;
Canterbury, Kent CT2 7NZ, United Kingdom;
Agronómica Nacional, 2780-157 Oeiras, Portugal; and
bDepartment of Biosciences, University of Kent,
cInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisbon, Avenida da República, Estação
dSchool of Chemistry, Newcastle University, Bedson Building, Newcastle upon Tyne NE1 7RU,
Edited by Rowena G. Matthews, University of Michigan, Ann Arbor, MI, and approved August 18, 2011 (received for review May 25, 2011)
Modified tetrapyrroles such as chlorophyll, heme, siroheme, vita-
min B12, coenzyme F430, and heme d1underpin a wide range of
essential biological functions in all domains of life, and it is there-
fore surprising that the syntheses of many of these life pigments
remain poorly understood. It is known that the construction of the
central molecular framework of modified tetrapyrroles is mediated
via a common, core pathway. Herein a further branch of the mod-
ified tetrapyrrole biosynthesis pathway is described in denitrifying
and sulfate-reducing bacteria as well as the Archaea. This process
entails the hijacking of siroheme, the prosthetic group of sulfite
and nitrite reductase, and its processing into heme and d1heme.
The initial step in these transformations involves the decar-
boxylation of siroheme to give didecarboxysiroheme. For d1heme
synthesis this intermediate has to undergo the replacement of
two propionate side chains with oxygen functionalities and the
introduction of a double bond into a further peripheral side chain.
For heme synthesis didecarboxysiroheme is converted into Fe-
coproporphyrin by oxidative loss of two acetic acid side chains.
Fe-coproporphyrin is then transformed into heme by the oxidative
decarboxylation of two propionate side chains. The mechanisms
of these reactions are discussed and the evolutionary significance
of another role for siroheme is examined.
enzymes ∣ metabolic pathway ∣ S-adenosylmethionine
process. Biochemical reactions are, in essence, highly controlled
and regulated chemical reactions that can also provide insights
into transformations for which no laboratory-based chemistry
may yet have been described. Such studies are at the heart of
chemical biology and are an essential prerequisite for its trans-
formation into synthetic biology. Moreover, from an evolutionary
perspective, there are many interesting questions relating to the
initial appearance of complex multistep pathways, where theories
on retro and patchwork models have been discussed (1, 2). These
considerations are pertinent to the synthesis of the modified
tetrapyrroles (3), which through their representatives chlorophyll,
heme, cobalamin (the biological form of vitamin B12), siroheme,
heme d1, and coenzyme F430are involved in a broad variety
of essential life processes from photosynthesis to methane pro-
The tetrapyrrolic architecture that underpins the structural
similarity between these various metalloprosthetic groups results
from a shared, though branched, biosynthetic pathway (4, 5),
where the whole family is derived from the macrocyclic primo-
genitor, uroporphyrinogen III (Fig. 1A). The pathways to heme
and chlorophyll via protoporphyrin have been previously char-
acterized. Similarly, the syntheses of siroheme and cobalamin
have also been elucidated, progressing via the bis-methylated
uroporphyrinogen III derivative precorrin-2 (Fig. 1A). However,
the biosynthesis of coenzyme F430and heme d1have not been
reported. Likewise, little is known about a proposed alternative
n understanding of biochemical pathways involves a compre-
hension of the chemical logic underpinning the synthetic
heme biosynthetic route that involves the transformation of
precorrin-2 into heme (6). This paper focuses on this unique
heme biosynthetic process and reveals a hitherto unsuspected
relationship with both the synthesis of siroheme and heme d1.
Siroheme, the prosthetic group of sulfite and nitrite re-
ductases, is synthesized from uroporphyrinogen III by bis-
methylation to give precorrin-2, dehydrogenation to produce
sirohydrochlorin and finally ferrochelation (7) (Fig. 1B). Heme
d1is only made by denitrifying bacteria with a cytochrome nitrite
reductase cd1(nirS) for which d1acts as an essential cofactor,
tailored to meet the mechanistic requirements of the reaction (8).
Bacteria with nirS always appear to contain, downstream of nirS,
a set of contiguous genes necessary for heme d1biogenesis (9). In
Paracoccus pantotrophus and Paracoccus denitrificans, these genes
are organized in the operon nirECFD-LGHJN (nirD and nirL
occur as a fused gene in Paracoccus species) and are transcribed
in the presence of nitric oxide (10). This operon is thought to
encode all the enzymes for heme d1biogenesis. So far only the
function of NirE has been unambiguously determined as an
S-adenosylmethionine (AdoMet)-dependent uroporphyrinogen
methyltransferase (11, 12), a finding that is consistent with pre-
vious labeling studies that demonstrate the synthesis of d1heme
must proceed via precorrin-2 (Fig. 1 A and C) (13).
Heme requires little introduction as a prosthetic group and
has a diverse range of biological functions. Its synthesis has been
elucidated in eukaryotes and most bacteria, where the classic
pathway for its biosynthesis is advanced via an ordered and se-
quential decarboxylation of the majority of the acidic side chains
of uroporphyrinogen III, followed by oxidation and ferrochela-
tion (4) (Fig. 1B). As alluded to earlier, in sulfate-reducing bac-
teria and Archaea, it is proposed that heme is made via a
completely different pathway, herein called the alternative heme
biosynthesis route. A combination of biochemistry and bioinfor-
matics approaches has provided some clues as to the mode of
operation of this route. Thus, feeding labeled methionine to
Desulfovibrio vulgaris cultures resulted in the isolation of labeled
sirohydrochlorin, 12,18-didecarboxysirohydrochlorin, copropor-
phyrin III and protoporphyrin IX (6). Similarly, analysis of com-
pletely sequenced Archaeal and Desulfovibrio genomes suggest
that these organisms possess the genes that encode enzymes for
the transformation of 5-aminolevulinic acid to uroporphyrinogen
but lack those required for the enzymes necessary for the conver-
sion of uroporphyrinogen into protoheme through the classical
Author contributions: S.B., A.D.L., S.A.L., L.M.S., S.J.F., and M.J.W. designed research;
S.B., A.D.L., S.A.L., D.J.P., and M.J.H. performed research; S.B., A.D.L., S.A.L., B.T.G., and
M.J.H. analyzed data; and S.B., L.M.S., B.T.G., S.J.F., and M.J.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/lookup/suppl/
18260–18265 ∣ PNAS ∣ November 8, 2011 ∣ vol. 108 ∣ no. 45www.pnas.org/cgi/doi/10.1073/pnas.1108228108
pathway (14–17). Altogether, these observations led to the theory
of an alternative heme biosynthesis pathway that diverges from
the classical pathway at the uroporphyrinogen step via precor-
rin-2 (Fig. 1 A and C).
Our genome comparison of denitrifying bacteria with some
sulfate-reducing bacteria and Archaea that make heme via pre-
corrin-2, revealed the presence of homologues of nirD, and nirJ
(see additionally ref. 17). The presence of these orthologues in
sulfate-reducing bacteria and Archaea, which do not need d1
heme as they do not have cd1, was intriguing because it implied
that they may have a role in the alternative heme biosynthesis
route. The similarity between the d1synthesis genes found in
Paracoccus and Desulfovibrio is shown diagrammatically in
Fig. 1D, where the alternative heme biosynthesis genes are given
the prefix ahb. Taken together, these observations prompted us to
investigate the link(s) between the alternate heme and d1heme
We speculated that the biosynthesis of heme and heme d1pro-
ceeds via the oxidized derivative of precorrin-2, sirohydrochlorin,
and didecarboxysirohydrochlorin, at which point the pathways for
the two cofactors would diverge. In both cases it was envisaged
that iron would be added at the final stage, as is observed with
siroheme and the classic heme pathway. To test the hypothesis
that heme d1synthesis may proceed via sirohydrochlorin, cell-free
extracts of Escherichia coli, overproducing either individual P.
pantotrophus Nir proteins (D-L, G, H, J, F) or combinations
of them generated using our link and lock technology (18), were
incubated with this metal-free substrate. Strikingly, when lysates
of E. coli, overproducing P. pantotrophus NirDL-G-H, were
incubated with sirohydrochlorin an instant change in the appear-
ance of the purple sirohydrochlorin to a blue-green color was
observed. The UV-visible spectrum of sirohydrochlorin, which
has a Soret band at 378 nm, also changed to a new maximum
around 390 nm (Fig. 2A). The shift of 12 nm in the Soret of
sirohydrochlorin and the disappearance of its d bands in the
region of 590 nm suggested that sirohydrochlorin had undergone
a chemical transformation and hence this unique compound was
NirDL-G-H Carries an Oxygen-Independent Siroheme Decarboxylase
Activity. HPLC analysis of the NirDL-G-H catalyzed conversion
of sirohydrochlorin showed the presence of two major peaks,
whose retention time and m∕z values differed from that of siro-
hydrochlorin (Fig. 2B). Significantly, the isotopic mass distribu-
biosynthesis. (A) Outline syntheses of modified tetrapyr-
roles highlighting key intermediates along the branched
pathway. Known reaction sequences are shown in black
whereas those not yet elucidated are highlighted in ma-
genta. (B) The known transformation of uroporphyrino-
gen III into siroheme and heme is shown. The numbering
system for the tetrapyrrole macrocycle is highlighted on
uroporphyrinogen III. (C) Precorrin-2 as the template for
both d1heme and heme synthesis is shown, with the lat-
ter derived via the alternative heme pathway. In both
cases the two methyl groups at C2 and C7, highlighted
in red, are derived from AdoMet. The enzymes involved
in the transformation of precorrin-2 into d1heme (Nir
enzymes) and heme (Ahb enzymes) are shown. (D) The
similarity between some of the Nir proteins implicated
in d1synthesis and proteins found in sulfate-reducing
bacteria and Archaea thought to be associated with
the Ahb pathway are indicated by black arrows.
Pathways and genes of modified tetrapyrrole
Bali et al.PNAS
November 8, 2011
tion pattern for the two compounds was consistent with the pre-
sence of iron. We surmised that sirohydrochlorin was converted
into siroheme via the capture of adventitious iron and then sub-
sequently underwent bis-decarboxylation into didecarboxysiro-
heme. To test this idea, siroheme was generated enzymatically
(trace D, Fig. 2B) and was found to have identical properties
to compound 1, observed in trace B, eluting at 15 min (Fig. 2B).
Moreover, when siroheme was added to cell lysates containing
NirD-L, G, and H, it was fully converted into didecarboxysiro-
heme (trace F, peak at 22 min). In contrast, when siroheme was
incubated with E. coli cell lysate overexpressing NirD-L, mono-
decarboxysiroheme was observed (trace E, peak at 18 min), sug-
gesting that the reaction occurs in a stepwise manner (Fig. 2B). In
control reactions where either sirohydrochlorin or siroheme were
incubated with the cell extracts of E. coli carrying empty expres-
sion vector-pET3a (Fig. 2B, traces B and D, respectively), no loss
of substrate or formation of didecarboxysiroheme was detected.
These results provide compelling evidence that the d1heme
biosynthesis pathway proceeds via siroheme and didecarboxysir-
oheme (Fig. 3). Although sequence analysis highlighted a signif-
icant sequence identity (35.3%) and similarity (47.1%) between
pairs of NirD-L, NirG, and NirH, these proteins are, on the basis
of mutagenesis, functionally nonredundant (9, 19).
sorption spectra of sirohydrochlorin (solid line) and didecarboxysiroheme
(dashed line), under anaerobic conditions in 50 mM potassium phosphate
buffer at pH 8. (B) HPLC traces of tetrapyrrole derivatives observed after
incubation of sirohydrochlorin (i–iii), and incubation of siroheme (iv–vi), with
the absorbance recorded at 390 nm. (B, i) HPLC trace of sirohydrochlorin.
(B, ii) HPLC trace of reaction containing E. coli cell lysate harboring empty
expression vector and sirohydrochlorin, note the new compound at 15 min.
(B, iii) HPLC trace of reaction containing NirD-LGH and sirohydrochlorin.
(B, iv) HPLC trace of siroheme. (B, v) HPLC trace of reaction containing
NirE,D-L and siroheme. (B, vi) HPLC trace of reaction containing NirD-LGH and
siroheme. Siroheme was used at a final concentration of 50 μM in each assay.
Siroheme decarboxylase activity of Nir proteins. (A) UV-visible ab-
modified tetrapyrrole pathway is shown with siroheme now acting as an
intermediate for heme and d1heme synthesis. This pathway can be com-
pared to Fig. 1C. (B) The branched pathway from siroheme to d1and heme
is shown together with the enzymes that are thought to be involved with
each particular step. Didecarboxysiroheme is generated from siroheme by
the sequential decarboxylation of the side chains attached to C12 and
C18. For d1synthesis, didecarboxysiroheme is modified by replacement of
the propionate side chains at C3 and C8 with oxo groups in a reaction likely
catalyzed by NirJ to generate a pre-d1intermediate, which is converted into
d1by the introduction of a double bond on the propionate side chain on C17.
For heme synthesis, didecarboxysiroheme is modified by the action of AhbC
to remove the acetic acid side chains attached to C2 and C7 to give Fe-copro-
porphyrin. This intermediate is converted into heme by AhbD through the
oxidative decarboxylation of propionate side chains on C3 and C8.
Deduced pathway from siroheme to d1and heme. (A) The corrected
www.pnas.org/cgi/doi/10.1073/pnas.1108228108Bali et al.
Subsequently, we found that extracts of E. coli overprodu-
cing NirD (where the C-terminal NirL encoding sequence was
removed from the plasmid) or NirH could catalyze a single dec-
arboxylation reaction of siroheme (Table S1). When extracts of
E. coli overproducing pairs of NirD-L or NirGH were incubated
anaerobically with siroheme, formation of a mixture of mono-
and didecarboxysiroheme was observed. However, for total con-
version of siroheme to didecarboxysiroheme all four proteins
were required (Table S1). The structure of didecarboxysiroheme,
arising from the loss of the carboxyl groups from the acetic acid
side chains attached to C12 and C18 in siroheme, was confirmed
by NMR (Fig. S1 and Table S2).
Siroheme is an Intermediate in both d1Heme and the Alternate Heme
Biosynthesis Pathway. To investigate if siroheme and didecar-
boxysiroheme are also intermediates in the alternative heme
biosynthesis pathway the homologous genes to nirDL, G, and H,
from both Desulfovibrio desulfuricans and D. vulgaris (ahbA-B),
were recombinantly produced in E. coli. When incubated with
siroheme, E. coli extracts containing AhbA and AhbB resulted
in the quantitative formation of decarboxylated siroheme
(Table S1). Moreover, an incubation of siroheme with purified
AhbA and AhbB resulted in the complete conversion of the
substrate into didecarboxysiroheme. Thus it would appear that
the decarboxylation of siroheme is also a committed step in the
biosynthesis of heme in sulfate-reducing bacteria and Archaea
that harbor these genes.
Siroheme is Converted into Heme in the Alternate Heme Biosynthesis
Pathway. Furthermore, incubation of anaerobically prepared
cell lysates of D. vulgaris with siroheme formed monodecarbox-
ysiroheme, didecarboxysiroheme, monovinyl Fe-coproporphyrin
III, and heme (Fig. S2). This observation is consistent with siro-
heme undergoing sequential decarboxylations to didecarboxy-
siroheme, then transformation into Fe-coproporphyrin by the
oxidative removal of the acetate side chains attached to C2
and C7 of the macrocycle and finally conversion into heme by the
sequential oxidative decarboxylation of the propionic acid side
chains attached to C3 and C8 (Fig. 3). All these reactions take
place under anaerobic conditions, and thus the final two steps
in this pathway are likely to involve some radical chemistry. Con-
sistent with this view is the observation that the remaining two
candidate genes identified in the alternative heme biosynthesis
pathway, ahbC and ahbD, both encode radical AdoMet enzyme
Role of AhbC and AhbD in Conversion of Didecarboxysiroheme to
Heme. To investigate the role of AhbC and AhbD, the two pro-
teins were recombinantly overproduced in E. coli. However,
aggregation problems with the D. desulfuricans AhbC resulted
in a search for a more soluble orthologue of the protein. Such
a candidate was found in Methanosarcina barkeri. Here the
M. barkeri AhbC was found to be recombinantly overproduced
in a soluble form in E. coli, and crude cell extracts containing
this protein were observed to transform didecarboxysiroheme
into Fe-coproporphyrin (Fig. 4). In contrast, the D. desulfuricans
AhbD was more amenable to purification and was isolated as
a brown solution with a broad peak between 340 to 400 nm in
the UV-visible spectrum, a feature typical for 4Fe-4S cluster-con-
taining proteins. This result is in agreement with the presence of
a conserved CX3CX2C motif in the primary sequence of AhbD
and its high homology to NirJ, the AdoMet-dependent radical
enzyme found in P. pantotrophus (20) (Fig. 1 and Fig. S3). When
a cell-free extract of E. coli overproducing D. desulfuricans
AhbD was incubated under strictly anaerobic conditions in the
presence of various cofactors for 4 h at room temperature
with Fe-coproporphyrin as substrate, heme, a monovinyl heme
derivative of Fe-coproporphyrin together with some unreacted
Fe-coproporphyrin were detected by liquid chromotagraphy
(LC)-MS (Fig. 5, trace B). In control reactions only background
levels of heme and unreacted Fe-coproporphyrin were detected
(Fig. 5, trace A and C). Finally, quantitative conversion of Fe-
coproporphyrin into heme occurred in extracts of E. coli with
D. vulgaris AhbD, AdoMet, and dithionite, thus confirming the
role of AhbD as a unique heme synthase.
The results presented herein have established a link between d1
heme and the alternative heme biosynthesis pathway, revealing
the surprising finding that both pathways proceed via siroheme
and then branch off at the didecarboxysiroheme stage (Fig. 3).
Incubations of heme d1biogenesis enzymes have led to the iden-
tification of siroheme as a substrate for d1synthesis and the
detection of an intermediate, didecarboxysiroheme, within the
pathway, providing unique insights into how the pathway must
operate. The enzyme complex required for the decarboxylation
of siroheme involves NirD-L, NirG, and NirH, proteins that are
classified as members of the Lrp/AsnC family (21), and recently
proposed to serve as regulators (22). In this respect, NirDL, G,
and H appear to compose a unique multifunctional enzyme and
transcriptional regulator. The reaction catalyzed by this enzyme
complex involves the decarboxylation of the acetic acid side
chains attached at C12 and C18 of the macrocycle. The mechan-
ism for such a reaction is likely to be similar to that utilized
by uroporphyrinogen decarboxylase, where an iminium ion is
Fe-coproporphyrin III. HPLC traces of the tetrapyrrole derivates observed
at 390 nm after anaerobic incubations of (A) E. coli cell-free extracts over-
expressing M. barkeri AhbC with didicarboxysiroheme (DDSH), AdoMet
and the reducing agent sodium dithionite, (B) E. coli cell-free extracts over-
expressing M. barkeri AhbC with DDSH and sodium dithionite, (C) E. coli
cell-free extracts with DDSH, AdoMet, and sodium dithionite. AdoMet and
sodium dithionite were used at final concentrations of 500 μM and
8.5 mM, respectively. Note that Fe-coproporphyrin III (Fe-Copro) is eluted
in two peaks from the HPLC column; both species have the same m∕z
value of 708 by MS.
M. barkeri AhbC catalyzes conversion of didicarboxysiroheme into
Bali et al.PNAS
November 8, 2011
generated to act as an electron sink (Fig. S4). Although we have
detected the presence ofa monodecarboxylated intermediate, the
order of decarboxylation has not been determined.
The biosynthesis of d1heme therefore proceeds via siroheme
and didecarboxysiroheme, and thereafter via a dioxo intermedi-
ate before the introduction of an acrylate function as the final
step. We believe that the oxo groups are formed by the action
of NirJ, a radical AdoMet enzyme (20), and that the double bond
in the propionate side chain attached to C17 may be mediated by
NirF, which, being periplasmic, must catalyze the last step. We
have shown that the periplasmic NirF is required for d1heme
production whereas two other periplasmic proteins (NirC and
NirN) coded for in the biogenesis operon are not (11, 23). Thus
we conclude that NirF catalyzes the final step in d1production
unless the product of NirF activity is imported to the cytoplasm
for further processing, an energetically expensive possibility. At
this stage we do not know what reaction is catalyzed by NirF
but the dehydrogenation to give the acrylate side chain of the iso-
bacteriochlorin is a possibility to be addressed in future work
(23). Our result that siroheme is an actual intermediate during
d1 heme biosynthesis means that a ferrochelatase activity is
not coded by the nir operon whose product must work in concert
with a siroheme synthesis pathway. It also shows that avoidance of
reactive radicals and potential toxicity by inserting iron at the end
of the biosynthetic pathway cannot be as important as previously
The similarity between the d1heme biosynthetic genes nirD-L,
G, and H and the putative alternative heme biosynthetic genes
ahbA and B immediately suggested that didecarboxysiroheme
could also be an intermediate in the latter pathway. This hypoth-
esis was confirmed by the activity of AhbA and AhbB upon
incubation with siroheme, which saw the quantitative conversion
into didecarboxysiroheme. Moreover, incubation of siroheme
with cell-free extracts of D. vulgaris resulted in the appearance
not only of didecarboxysiroheme but also Fe-coproporphyrin
and heme [and it is also interesting to note that Fe-coproporphyr-
in is found as a cofactor in the bacterioferritin from D. desulfur-
icans (24)]. Thus, heme synthesis would appear to be mediated
by the conversion of siroheme into didecarboxysiroheme, fol-
lowed by oxidative loss of the northern acetic acid side chains
and then oxidative decarboxylation of the northern propionate
side chains to vinyl groups (Fig. 3).
The loss of the acetic acid side chains at C2 and C7 for the
synthesis of Fe-coproporphyrin is catalyzed by AhbC, which is
a radical AdoMet family member. To promote this reaction
the enzyme is likely to generate adenosyl radicals and use these
to abstract hydrogen atoms in turn from the C3 or C8 positions
thereby producing the respective substrate radical and deoxyade-
nosine. The radical can then fragment by homolytic cleavage
of the C-C bond connecting the side chain acetic acid group to
the macrocycle, generating a double bond and an acetate radical
(Fig. S5A). This process is analogous to the proposed fragmenta-
tion of glutamate in the reaction catalyzed by coenzyme B12-de-
pendent glutamate mutase, where an acrylate molecule and glycyl
radical are formed prior to the rearrangement into 3-methylas-
partate (25, 26). However, in the case of the reaction catalyzed
by AhbC the enzyme presumably converts the acetate radical to
acetate by providing a further electron (and proton), which could
come from a second predicted Fe-S center on the protein. The
mechanism as described consumes two molecules of AdoMet,
each of which generates an adenosyl radical. Alternatively, an
initially produced adenosyl radical could be recycled by using
the methyl group of deoxyadenosine as a source of a hydrogen
atom to convert the acetate radical into acetate.
The final reaction in the alternative heme biosynthesis path-
way, catalyzed by AhbD, sees the transformation of Fe-copropor-
phyrin into heme. Overall, this process is similar to the HemN
catalyzed oxidative decarboxylation reaction of coproporphyrino-
gen III to protoporphyrinogen IX that occurs during the classical
heme biosynthesis pathway (27, 28). As with HemN (and AhbC),
AhbD is also a radical AdoMet family member. By analogy with
HemN, the mechanism of AhbD is likely to involve the abstrac-
tion of a hydrogen atom from the beta-position of the propionate
side chains attached to C3 and C8. The resulting substrate radical
can then further oxidize to a carbon cation, which could lead to
the vinyl product by loss of CO2(Fig. S5B).Although a monovinyl
intermediate has been identified, it is not known if there is a pre-
ferential order for the two oxidative decarboxylations.
The identification of the intermediates of both the heme and
heme d1synthesis pathways highlights some of the ingenious
chemistry that takes place. These beguiling reactions include
the incorporation of oxo functionalities in place of the northern
propionate side chains during d1synthesis from didecarboxysir-
oheme and the oxidative loss of the northern acetate side chain
from the same branch-point intermediate during heme synthesis.
The sequence similarity between NirJ and AhbC not only reflects
the proteins being members of the radical AdoMet family but
may also reflect recognition of the same substrate. The assign-
ment of function to these enzymes will now permit a more de-
tailed mechanistic investigation into how these transformations
Our research also defines a fresh role for siroheme, previously
only thought to act as a prosthetic group for assimilatory sulfite
and nitrite reductases, as an intermediate in modified tetrapyr-
role biosynthesis (Fig. 3A). From an evolutionary perspective,
it has previously been suggested that siroheme may have acted
as a primordial heme where the isobacteriochlorin ring would
have allowed greater conformational flexibility, prior to the selec-
tion of porphyrins in the later more oxidizing environments (29).
There is a view that denitrification came early in evolution and
it is possible that the d1-containing nitrite reductase appeared
before both oxygen and the copper nitrite reductase (30). This
paper now highlights how heme can be synthesized from siro-
heme, providing a biosynthetic evolutionary corollary relation-
ship whereby the functional evolution of siroheme into heme is
linked by the appearance of a biochemical pathway allowing
the physical transformation of one into the other. The seemingly
observed porphyrin derivatives at 390 nm in reaction containing, (A) Fe-co-
proporphyrin, cofactor mix, and cell-free extract of E. coli harboring empty
expression vector, (B) Fe-coproporphyrin, cofactor mix, and cell-free extract
of E. coli overexpressing D. desulfuricans AhbD, and (C) Fe-coproporphyrin,
cell-free extract containing D. desulfuricans AhbD without AdoMet in the
cofactor mix. Cofactor mix consisted of 500 μM AdoMet and NADPH with
0.3% (vol∕vol) Triton X-100 in each assay. Fe-coproporphyrin was used at
a final concentration of 25 μM, and is eluted as two close peaks from the
HPLC column but with same m∕z value of 708 by MS.
AhbD catalyzed Fe-coproporphyrin oxidase activity. HPLC traces of
www.pnas.org/cgi/doi/10.1073/pnas.1108228108 Bali et al.
coincidental molecular hijacking of the cofactor for assimilatory
nitrite reductase to form the cofactor for dissimilatory nitrite
reductase was unanticipated; we cannot rule out the reverse
appearance first of d1heme followed by the hijacking of siro-
heme by the assimilatory nitrite reductase, which might have
emerged once oxidized nitrogen increased as atmospheric oxygen
increased. The presence of this branch of tetrapyrrole biosynth-
esis is consistent with a patchwork assembly model of pathway
evolution, and provides a hitherto unsuspected relationship
between nitrite assimilation and dissimilation.
DNA Manipulations. DNA manipulations were performed by standard meth-
ods. Amplifications of genes from P. pantotrophus (GB17), D. desulfuricans
(G20), and D. vulgaris Hildenborough were performed by PCR using KOD
DNA polymerase (from Thermococcus kodakaraensis) according to supplier’s
instructions (Novagen). All constructs generated by PCR were confirmed to
be correct by sequencing. Plasmids containing nirED-L, nirGH, and nirD-
LGH were constructed by inserting the appropriate amplified P. pantotrophus
genes into pET3a using the link andlock method described in ref. 18. ThenirH
was cloned into pET14b, and thus the recombinant protein contained an
N-terminal hexahistidine tag, whereas nirG and nirD-L were cloned into
pET3a and pASKIBA13 plus vectors, respectively. Plasmids containing ahbA
and ahbB from D. desulfuricans were obtained by cloning into the vectors
pKK2233 and pET14b, respectively, whereas a plasmid harboring ahbAB from
D. vulgaris was obtained by cloning the genes into pET3a vector using the
link and lock method described above.
Recombinant Protein Production. Cell growth and protein overexpression. For
protein overproduction, the E. coli BL21(DE3) strain was transformed with
the appropriate plasmid. The resulting strain was grown with aeration at
37 °C in LB medium containing appropriate antibiotics (50 μg∕mL ampicillin,
30 μg∕L chloramphenicol, to an A6000.6–0.8 and the protein expression was
induced with 0.4 mM IPTG and cultures were transferred to 16 °C cells for
overnight expression. Cells were collected by centrifugation 3;500 × g for
15 min at 4°C (Beckman Coulter, JI30). The cell pellets from 1 L of LB broth
were resuspended in 20 mL of 50 mM Tris·HCl, pH 8.0, containing 0.5 M NaCl.
Cells were disrupted by sonication (Sonics Vibracell Ultrasonic processor). Cell
debris and insoluble proteins were removed from the soluble cell lysate by
centrifugation 35;000 × g for 20 min at 4°C. A cell extract of sulfate-reducing
bacteria was obtained from D. vulgaris Hildenborough frozen cell pellets.
Purification of 5-aminolevulinic acid and succinyl-CoA synthases. Recombinant
Rhodobacter sphaeroides 5-aminolevulinic acid synthase (HemA) was pro-
duced and purified as described in ref. 28. Recombinant E. coli succinyl-coen-
zymeA synthetase (SucCD) was purified as a hexahistidine fusion protein as
follows. Cell lysate containing the overexpressed SucCD was applied to the
Ni-Sepharose column equilibrated in buffer A (0 mM Tris·HCl, pH 8.0 buffer).
The column was washed with 5–10 column volumes of 50 mM imidazole con-
taining buffer A and the proteins were eluted using the 400 mM imidazole
containing buffer A. Only SucC (41,400 Da) has a hexahistidine tag but the
associated SucD (30,000 Da), which was not tagged, also eluted.
Purification of multienzyme cocktail for sirohydrochlorin production. For in
vitro preparation of sirohydrochlorin a multienzyme method was used as
described previously (15) and outlined briefly in SI Text and Fig. S6.
Production of siroheme. To make siroheme from sirohydrochlorin, a 10-fold
excess of FeSO4was added to a sirohydrochlorin solution and the mixture
was left at room temperature for 4 h before purification on the DEAE
column. The siroheme was purified on a small DEAE column to assist NMR
studies with the siroheme and the didecarboxysiroheme derivative.
High performance LC-MS analysis of siroheme and siroheme derivatives.
The tetrapyrrole intermediates could be purified after separation from
the protein by boiling at 80°C for 10 min and were analyzed by reverse phase
chromatography. Samples were resolved on an ACE 5AQ column (2.1×
150 mm, 5 μ, Advanced Chromatography Technologies) attached to a Agilent
1100 series HPLC equipped with diode array detector and coupled to a
micrOTOF-Q (Bruker) mass spectrometer. The column was developed with
a binary gradient at a flow rate of 0.2 mL min−1. Solvent A was 0.1% TFA
and solvent B was acetonitrile. The column was equilibrated with 5% B.
Following sample injection the concentration of B was increased to 20%
over 6 min and then to 30% at 25 min and 100% at 35 min where it was
held for 5 min before returning to starting conditions. The total length of
each run was 50 min. Alternatively, for incubations starting from Fe-copro-
porphyrin III a faster linear gradient was employed starting at 20% B and
reaching 100% B in 30 min. The observed masses for these various intermedi-
ates are given in SI Text and shown in Fig. S7.
ACKNOWLEDGMENTS. We thank Dr. Michelle Rowe for technical support.
This work was supported by Biotechnology and Biological Sciences Research
Council Grants BBE0229441 and BB/E024203 (to S.J.F and M.J.W.) and
Wellcome Trust Equipment Grant 091163/Z/10/Z (to M.J.H. and M.J.W.).
1. Horowitz NH (1945) On the evolution of biochemical syntheses. Proc Natl Acad Sci USA
2. Jensen RA (1976) Enzyme recruitment in evolution of new function. Annu Rev Micro-
3. Holliday GL, et al. (2007) Evolution of enzymes and pathways for the biosynthesis of
cofactors. Nat Prod Rep 24:972–987.
4. Layer G, Reichelt J, Jahn D, Heinz DW (2010) Structure and function of enzymes in
heme biosynthesis. Protein Sci 19:1137–1161.
5. Warren MJ, Raux E, Schubert HL, Escalante-Semerena JC (2002) The biosynthesis of
adenosylcobalamin vitamin B12. Nat Prod Rep 19:390–412.
6. IshidaT, et al.(1998) A primitive pathwayof porphyrin biosynthesisand enzymologyin
Desulfovibrio vulgaris. Proc Natl Acad Sci USA 95:4853–4858.
7. Raux E, et al. (2003) Identification and functional analysis of enzymes required for
precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem
and cobalamin in Bacillus megaterium. Biochem J 370:505–516.
8. Rinaldo S, et al. (2011) Observation of fast release of NO from ferrous d1haem allows
formulation of a unified reaction mechanism for cytochrome cd1nitrite reductases.
Biochem J 435:217–225.
9. Palmedo G, et al. (1995) Resolution of the nirD locus for heme d1synthesis of cyto-
chrome cd1(respiratory nitrite reductase) from Pseudomonas stutzeri. Eur J Biochem
10. Zajicek RS, Ferguson SJ(2005) The enigmaof Paracoccuspantotrophuscytochromecd1
activation. Biochem Soc Trans 33:147–148.
11. Storbeck S, et al. (2009) The Pseudomonas aeruginosa nirE gene encodes the
S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase required
for heme d1biosynthesis. FEBS J 276:5973–5982.
12. Zajicek RS, et al. (2009) d1haem biogenesis—assessing the roles of three nir gene
products. FEBS J 276:6399–6411.
13. Yapbondoc F, Bondoc LL, Timkovich R, Baker DC, Hebbler A (1990) C-methylation
occurs during the biosynthesis of heme-d1. J Biol Chem 265:13498–13500.
14. Cavallaro G, Decaria L, Rosato A (2008) Genome-based analysis of heme biosynthesis
and uptake in prokaryotic systems. J Proteome Res 7:4946–4954.
15. Lobo SAL, Brindley A, Warren MJ, Saraiva LM (2009) Functional characterization of
the early steps of tetrapyrrole biosynthesis and modification in Desulfovibrio vulgaris
Hildenborough. Biochem J 420:317–325.
16. Panek H, O’Brian MR (2002) A whole genome view of prokaryotic haem biosynthesis.
17. Storbeck S, et al. (2010) A novel pathway for the biosynthesis of heme in Archaea:
Genome-based bioinformatic predictions and experimental evidence. Archaea
18. McGoldrick HM, et al. (2005) Identification and characterization of a novel vitamin B12
(cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing
flavin, heme, and Fe-S cofactors. J Biol Chem 280:1086–1094.
19. Kawasaki S, Arai H, Kodama T, Igarashi Y (1997) Gene cluster for dissimilatory nitrite
reductase (nir) from Pseudomonas aeruginosa: Sequencing and identification of a
locus for heme d1biosynthesis. J Bacteriol 179:235–242.
20. Brindley AA, Zajicek R, Warren MJ, Ferguson SJ, Rigby SE (2010) NirJ, a radical SAM
family member of the d1heme biogenesis cluster. FEBS Lett 584:2461–2466.
21. Xiong J, Bauer CE, Pancholy A (2007) Insight into the haem d1biosynthesis pathway
in heliobacteria through bioinformatics analysis. Microbiology-Sgm 153:3548–3562.
22. Oglesby-Sherrouse AG, Vasil ML (2010) Characterization of a heme-regulated non-
coding RNA encoded by the prrF Locus of Pseudomonas aeruginosa. PLoS One
23. Bali S, Warren MJ, Ferguson SJ (2010) NirF is a periplasmic protein that binds d1heme
as part of its essential role in d1heme biogenesis. FEBS J 277:4944–4955.
24. Romao CV, et al. (2000) Iron-coproporphyrin III is a natural cofactor in bacterioferritin
from the anaerobic bacterium Desulfovibrio desulfuricans. FEBS Lett 480:213–216.
25. Buckel W, Kratky C, Golding BT (2005) Stabilization of methylene radicals by cob(II)
alamin in coenzyme B12dependent mutases. Chemistry Eur J 12:352–362.
26. Marsh ENG, Patterson DP, Li L (2010) Adenosyl radical: Reagent and catalyst in enzyme
reactions. ChemBioChem 11:604–621.
27. Layer G, Moser J, Heinz DW, Jahn D, Schubert WD (2003) Crystal structure of
coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes.
EMBO J 22:6214–6224.
28. Layer G, et al. (2006) The substrate radical of Escherichia coli oxygen-independent
coproporphyrinogen III oxidase HemN. J Biol Chem 281:15727–15734.
29. Crane BR, Getzoff ED (1996) The relationship between structure and function for
the sulfite reductases. Curr Opin Struct Biol 6:744–756.
30. Ducluzeau AL, et al. (2009) Was nitric oxide the first deep electron sink? Trends
Biochem Sci 34:9–15.
Bali et al. PNAS
November 8, 2011