Oxygen-independent decarbonylation of aldehydes by cyanobacterial aldehyde decarbonylase: A new reaction of diiron enzymes

Article abstract of DOI:10.1002/anie.201101552

Just add water: Structurally, cyanobacterial aldehyde decarbonylases are members of the non-heme diiron oxygenase family of enzymes. However, the enzyme catalyzes the hydrolysis of aliphatic aldehydes to alkanes and formate (see scheme), in an oxygen-independent reaction. This unusual and chemically difficult reaction most likely involves free radical intermediates. Copyright

Full text of DOI:10.1002/anie.201101552

Communications
DOI: 10.1002/anie.201101552
Enzymology
Oxygen-Independent Decarbonylation of Aldehydes by
Cyanobacterial Aldehyde Decarbonylase: A New Reaction of Diiron
Enzymes**
Debasis Das, Bekir E. Eser, Jaehong Han, Aaron Sciore, and E. Neil G. Marsh*
The search for new biofuels has generated increased interest
in biochemical pathways that produce hydrocarbons.[1]
Although hydrocarbons are simple molecules, the biosynthe-
sis of molecules that lack any chemical functional groups is
surprisingly challenging.[2] Biochemical reactions that remove
functionality, such as decarboxylations, dehydrations and
reduction of double bonds, invariably rely on the presence of
adjacent functional groups to stabilize unfavorable transition
states. Enzymes involved in hydrocarbon biosynthesis are
therefore of interest both for applications in biofuels pro-
duction and because of the unusual and chemically difficult
reactions they catalyze.[3] One enzyme that has attracted
particular interest, is aldehyde decarbonylase (AD), which
catalyzes the decarbonylation of long-chain fatty aldehydes,
to the corresponding alkanes.[4]
dinuclear iron oxygenase family of enzymes exemplified by
methane monoxygenase, type I ribonucleotide reductase, and
ferritin.[9] In all these enzymes, the diiron center is contained
within an antiparallel four-a-helix bundle in which two
histidines and four carboxylates (either from aspartate or
glutamate) supply the protein ligands to the metal ions. The
dinuclear iron center of cAD superimposes very closely on
those of the other enzymes; however in the structure the
carboxylate of an adventitiously bound long-chain fatty acid
bridges the two iron atoms, displacing one of the glutamate
ligands (Figure 1).
AD was originally identified in studies on the biosynthesis
of hydrocarbon waxes by higher plants and algae.[5] These
enzymes are integral membrane proteins of Mr ꢀ 70000 Da
that convert long-chain aldehydes, derived from fatty acids, to
alkanes and carbon monoxide. The enzymes require divalent
metal ions for activity;[4b,6] however, the identity of the metal
remains unclear. It was reported that the pea enzyme is active
with Co, Cu, or Zn[6] whereas the Botryococcus braunii
enzyme uses Co.[4b] Sequence analysis of cer1, a gene encoding
a putative AD from Arabidopsis thaliana, indicates that it is
member of the sterol oxidase class of non-heme iron-
dependent oxygenases.[7]
More recently, a soluble version of AD was discovered in
cyanobacteria (cAD).[4c] Serendipitously, a crystal structure
for cAD from Prochlorococcus marinus MIT9313 had
previously been solved as part of a structural proteomics
project, although no function had been assigned.[8] The
structure revealed that cAD is member of the non-heme
Figure 1. Structure of cAD from P. marinus (PDB ID 2OC5A).
a) Ribbon diagram showing position of iron atoms and co-crystallized
fatty acid. b) Structure of the diiron cluster showing protein ligands
and co-crystallized fatty acid which displaces one Glu ligand. c) Com-
parison with the diiron cluster of methane monooxygenase from
Methylococcus capsulatus (Bath) (PDB ID 1XVC).
[*] D. Das,[+] Dr. B. E. Eser,[+] A. Sciore, Prof. E. N. G. Marsh
Department of Chemistry, University of Michigan
Ann Arbor, MI 48109 (USA)
Fax: (+1)734-615-3790
E-mail: nmarsh@umich.edu
Prof. J. Han
School of Biological Sciences, Chung-Ang University
Anseong 456-756 (Korea)
[+] These authors contributed equally to this work.
The reactions catalyzed by this structural class of diiron
enzymes invariably involve molecular oxygen[9] and typically
require an external reducing system in the form of ferredoxin,
ferredoxin oxidoreductase and NADPH, to reduce the
“resting” diferric form of the enzyme to the active diferrous
form at the start of each turnover. It was established that cAD
also requires this external reducing system to catalyze the
conversion of aldehydes to alkanes.[4c]
[**] This work was supported in part by grants from the American
Chemical Society Petroleum Research Fund 48781 ND4, European
Union FP-7 256808, and the National Institutes of Health GM
093088 to E.N.G.M.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201101552.
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Most recently it was shown that, rather than producing
CO as a co-product, cAD converts aldehydes to alkanes and
formate.[10] Formally this appears to be a hydrolysis reaction,
albeit a very unusual one. However, it was found that, like
other members of diiron oxygenase family, cAD utilizes
molecular oxygen and that one atom of oxygen is incorpo-
rated into formate.[11] This led the authors to propose that
cAD catalyzes a “cryptic” oxidation reaction (Scheme 1) in
tion; the activity of other metals was indistinguishable from
background activity.
We also investigated whether a hetero metal cluster may
support activity, or promote enhanced activity. Assays were
conducted in which the molar ratio of FeII to the second metal
ion was varied between 0 and 100% and cAD activity plotted
as function of iron percentage (Figure S4). Mn, Co, Cu, and Ni
had no effect on activity whereas Zn appeared to be slightly
inhibitory. Thus cAD appears to function as a dinuclear iron
enzyme.
Having established that cAD is iron-dependent, we
investigated the kinetics and cofactor requirements for the
production of alkanes by the enzyme. Although the con-
version of n-octadecanal to n-heptadecane by cAD was
established in the initial identification of the enzyme,[4c] no
kinetic information was reported. We confirmed that hepta-
decane formation was absolutely dependent upon the pres-
ence of ferredoxin, ferredoxin oxidoreductase, and NADPH.
However, based on assay conditions reported by Schirmer
et al.,[4c] that is, 5 mm cAD, 10 mm ferrous ammonium sulfate,
300 mm octadecanal, 30 mgmLÀ1 spinach ferredoxin, 0.04
UmLÀ1 ferredoxin reductase and 800 mm NADPH, the
reaction proceeded extremely slowly and typically only
about 1 turnover could be achieved in 1 h.
During the course of these initial investigations, we
rapidly established that cAD is fully active under strictly
anaerobic conditions (Figure S5) and thus does not require
oxygen for activity. Indeed, activity was approximately 40%
higher than in the presence of oxygen. Therefore, subsequent
studies were conducted anaerobically. This observation is
particularly surprising given that all other diiron enzymes in
this structural family catalyze reactions involving O2, and the
recent observation that cAD incorporates oxygen into
formate under aerobic conditions.[11] Clearly a “cryptic”
oxidation mechanism cannot operate anaerobically and so
the reaction must occur by a fundamentally different mech-
anism.
Because of the poor activity observed with the ferredoxin
system, we investigated other biochemical reducing systems.
We discovered that the commonly used 5-methylphenazinium
methylsulfate (PMS):NADH reducing system[14] could effec-
tively substitute for the ferredoxin system, and resulted in
greatly improved activity. Replacing the ferredoxin system
with 75 mm PMS and 750 mm NADH in the above assay
allowed reaction rates to be determined from the initial time
points, with turnover numbers approaching 0.4 minÀ1 at 378C.
The reaction rate increased linearly with increasing octade-
canal concentration and showed no sign of saturation at
concentrations up to 500 mm (Figure 2). An apparent kcat/Km
of ca. 1 minÀ1 mmÀ1 may be calculated from these data, but as
discussed below this probably significantly underestimates
the true value of kcat/Km.
Scant kinetic data has been reported for the oxygen-
dependent reaction of cAD; Warui et al. estimated that only
around 3 turnovers had occurred in 20 h.[10] This probably
underestimates the rate of the oxygen-dependent reaction,
but based on this number cAD is 1000 to 100-fold more active
in the anaerobic reaction with the PMS:NADH reducing
system.
Scheme 1. “Cryptic” oxidation of aldehydes to alkanes and formate
proposed for the oxygen-dependent reaction of cAD.
which an iron peroxo species attacks the carbonyl group to
À
facilitate the chemically difficult scission of the C1 C2 bond;
finally, further reduction of oxygen was proposed to occur so
that overall O2 is reduced to water.
Here we report that cAD catalyzes the decarbonylation of
aldehydes under anaerobic conditions to give the same
products: that is, an alkane and formate. Moreover, this
reaction occurs at rates that are 100–1000 times higher than
those reported for the oxygen-dependent reaction. We also
present evidence that the aldehyde substrate interacts with
metal site and demonstrate, for the first time, that iron is the
active metal in cAD. Intriguingly, even under anaerobic
conditions a reducing system is still required. Overall, our
results point to a new and fundamentally different mode of
reactivity for non-heme diiron enzymes.
The experiments we report were conducted on cAD from
Prochlorococcus marinus MIT9313. The enzyme was over-
expressed in E. coli from a synthetic gene, codon-optimized
for expression in E. coli, and purified by standard methods
(see the Supporting Information).
Given the very unusual nature of the reaction, and that
several different metal ions have been reported as necessary
for AD activity, we considered it essential to establish the
metal requirements for cAD. In particular, it has been
speculated that cAD may be a Mn-requiring enzyme,
analogous to the Mn/Fe-dependent ribonucleotide reducta-
ses.[4c,10–12] The enzyme, as isolated, was pale brown and the
UV/Vis spectrum exhibited a shoulder at 350 nm character-
istic of ferric iron (Figure S3 in the Supporting Information).
Metal analysis by ICP-MS established that in a typical
preparation only 30–35% of the purified enzyme contained
metal ions (assuming 2 metal ions per protein). The following
metal composition represents that found in one enzyme
preparation: Fe 17.8%, Zn 9.6%, Ni 5.1%, Mn 3.0%, Cu
< 0.2%, Co < 0.1%.
Endogenous metals could be removed from cAD by first
incubating the enzyme with 10 mm ferrozine and 5 mm
sodium dithionite overnight,[13] followed by dialysis against
buffers containing 10 mm EDTA (ethylenediaminetetraacetic
acid) and 10 mm NTA (nitrilotriacetic acid). This allowed the
apoenzyme to be reconstituted with biological relevant
divalent metal ions, including Mn, Fe, Co, Ni, Cu and Zn,
and their activity tested. Only FeII supported alkane forma-
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formate results from a side-reaction. Attempts to detect CO
formation by cAD, either by using myoglobin to bind CO[16]
(Figure S6) or by MS analysis of the reaction head space, were
all negative.
When the reaction was performed in D2O, the product
alkane contained deuterium, the labeling pattern being
independent of whether the substrate was deuterated (Fig-
ure 3b). (Some additional deuterium derives from exchange
of the a-protons of the aldehyde with solvent during the
reaction; however, control experiments established that this
accounts for less than half of the deuterium incorporation.)
Whereas when the reaction was performed with deuterated
octadecanal the aldehyde proton was retained in formate, as
determined by MS analysis of the 2-NPH derivative (Fig-
ure 3c).
Figure 2. Kinetics of heptadecane formation by cAD. a) Time course of
heptadecane formation (octadecanal concentration=200 mm); b) rate
of reaction as a function of substrate concentration.
The low rates of catalysis exhibited by cAD undoubtedly
derive in part from the very poor solubility of the substrate.
Addition of detergents such as Triton did not improve activity
noticeably. Assays had to be shaken vigorously to obtain
reproducible results. Even at low concentrations the substrate
appears to be present as micelles in solution; and it is very
likely that reaction kinetics are dominated by phase transfer
of substrate molecules from micelles. Furthermore, the
ferredoxin system seemed to be inactivated by continuous
shaking, which may explain the poor activity observed in
using this reducing system.
Thus it appears that under anaerobic conditions the
conversion of aldehydes to alkanes and formate catalyzed by
cAD is a true hydrolysis reaction, as shown in Scheme 2.
We note that Li et al.[11] reported that cAD was inactive in
the absence of O2. The reason for the discrepancy with our
results is unclear. However, they used cAD from a different
cyanobacterium, Nostoc punctiforme, which has an approx-
imately 60% sequence identity with the P. marinus enzyme.
Sequence differences could conceivably alter substrate bind-
ing in the active site. Their experiments were also conducted
under different assay conditions, with a reducing system that
supports much lower activity to start with.
We established that the anaerobic reaction of cAD with
octadecanal also produces formate. Formate was confirmed as
the co-product by derivatizating the products of reaction with
2-nitrophenyl hydrazine (2-NPH)[15] and subsequent analysis
by reverse-phase HPLC and mass spectrometry (Figure 3c).
GC analysis of heptadecane from the same reaction con-
firmed that formate and heptadecane were produced stoi-
chiometrically during the reaction, making it unlikely that
Scheme 2. Oxygen-independent hydrolytic conversion of aldehydes to
alkanes and formate catalyzed by cAD.
Given that the cAD reaction does not require O2, and is
redox neutral, we reconsidered the role of the external
reducing system in the reaction. For most iron-dependent
oxygenases the reducing system functions to reduce FeIII to
FeII, which then reacts with oxygen.[9e,17] In aerobic conditions
the reducing system may function to reduce the ferric form of
cAD to the ferrous form. However, if this were the only
function of the reducing system it should not be required
under anaerobic conditions. To test this, we examined the
activity of apo-cAD reconstituted with FeII under rigorously
anaerobic conditions in the absence of external reductant.
Reactions containing 100 mm apo-cAD reconstituted with
Figure 3. Identification of the products of the cAD reaction. a) GC-MS chromatograph demonstrating the conversion of octadecanal to
heptadecane; b) mass spectra of heptadecane demonstrating that hydrogen derives from the solvent and not from the aldehyde proton; c) mass
spectra of recovered 2-NPH-derivatized formate demonstrating that the aldehyde proton or deuteron is retained in formate.
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200 mm FeII were incubated with 200 mm octadecanal for
periods of up to 24 h. However, no heptadecane formation
could be detected. (The limit of detection in the experiment
was < 1 mm heptadecane.) Thus an external reducing system
appears to be absolutely required for the formation of
alkanes. This strongly suggests that it performs a catalytic
role in the reaction rather than simply being required to
maintain the enzyme in the ferrous state.
By analogy with the reaction of diiron enzymes with O2,
we hypothesized that the first step in the cAD reaction may
involve electron transfer from FeII to the carbonyl group of
the aldehyde. To test this, we investigated the interaction of
the substrate with the diiron center by EPR spectroscopy
(Figure 4). The apo-enzyme was reconstituted with FeII under
This observation is consistent with FeII transferring an
electron to the substrate to generate FeIII and, presumably, a
ketyl radical that is too unstable to observe directly. Further
support for the generation of an organic radical came from
experiments in which the spin-trapping agent N-tert-butyl-a-
phenylnitrone (phenyl-N-tert-butylnitrone, PBN), which has
been used to trap radical intermediates in other enzyme
reactions,[19] was included in the reaction. The ferric signal is
now accompanied by a characteristic signal for the PBN
nitroxide radical adduct at g = 2. The hyperfine structure of
the nitroxide radical is broadened, indicating that it results
from PBN intercalated within the protein,[19] rather than free
in solution. This is consistent with PBN reacting with the
substrate radical, or possibly a protein-based radical, as it is
formed at the active site of the enzyme. Addition of PBN in
the absence of heptanal (Figure 4d) appears to result in some
oxidation of FeII, but very little radical adduct is observed.
Although many details of this remarkable reaction remain
to be elucidated, we wish to propose a tentative mechanism
that accounts for the experimental observations above
(Scheme 3). Our results provide evidence that in the first
Figure 4. EPR spectra of cAD prepared under anaerobic conditions:
a) apo-cAD reconstituted with FeII to form diferrous enzyme; b) addi-
tion of heptanal to diferrous cAD; c) addition of heptanal and spin
trapping reagent PBN to diferrous cAD (inset: expansion of g=2
region of this spectrum showing PBN radical adduct); d) addition of
PBN only to diferrous cAD; e) spectrum of diferrous enzyme ion at 6 K
(20 mW microwave power). All spectra except (e) were recorded at
9.395 GHz, microwave power 2 mW, modulation amplitude 16 G,
temperature 77 K, and are plotted on the same scale.
Scheme 3. A mechanistic proposal for cAD that accounts for the
reported experimental observations.
step aldehyde binding to the diferrous iron center initiates
electron transfer from iron to the substrate. This would result
in an iron-bound ketyl radical, 2, analogous to the superoxo
species formed by other diiron enzymes in their reaction with
O2. At this point, injection of an additional electron from the
reducing system would serve both to reduce the iron cluster,
and to generate the more reactive ketyl radical anion, 3. We
anaerobic conditions (holoenzyme concentration 425 mm) and
spectra were recorded at 77 K and at 6 K. Experiments were
conducted in the absence of the reducing system so that
turnover could not occur. At 77 K the enzyme is EPR-silent,
as expected for a diferrous iron center,[18] however, at 6 K the
presence of the diferrous center is confirmed by a broad, weak
resonance centered at g = 13. Addition of heptanal (600 mm)
to holo-cAD resulted in the appearance of a signal at g = 4.3
characteristic of a high-spin ferric ion. (Heptanal, rather than
octadecanal, was used in this experiment due to its higher
solubility. Heptanal is a substrate for cAD, see the Supporting
Information.)
À
then propose that 3 undergoes homolytic cleavage of the C1
C2 bond to form a methylene radical and that concomitantly
the formyl group becomes covalently bound to iron (4).
Proton-coupled electron transfer (with water or a protein
side-chain supplying the proton) would result in alkane
formation (5) and a ferric hydroxyl species that could serve to
hydrolyze the iron–formyl complex. Lastly, return of an
electron to the reducing system and hydrolysis of the iron–
formyl complex to give formate and regenerate the ferrous
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cluster, 6, must occur. The precise mechanism and timing of
these latter steps must remain more speculative.
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À
precedent for C C scission by a ketyl radical, similar species
À
have been shown to facilitate C O cleavage in hydroxyglu-
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clocks” such as cyclopropyl rings.[22] Iron–formyl complexes
have been characterized in diiron compounds that model the
hydrogenase active site, although these are stabilized by CO
ligands.[23] We note that transiently reducing the iron center
during the reaction would stabilize the formation of the
proposed iron–formyl complex (intermediates 4 and 5), and in
thereby facilitate decarbonylation. It might also allow these
intermediates to be trapped at low temperatures by rapid
freeze–quench methods to permit their spectroscopic charac-
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In conclusion, it appears that cAD can catalyze the same
reaction by two quite different mechanisms. The oxygen-
independent decarbonylation of aldehydes catalyzed by cAD
represents a radical departure from the “conventional”
oxygen activation chemistry catalyzed by diiron oxygenase
enzymes. The enzyme appears to exploit the reduced diiron
center to facilitate novel radical chemistry that requires
electron transfer from an external reductant. The hydrolysis
of aldehydes in this manner presents a formidable challenge
À
because breaking the C1 C2 bond either heterolytically, to
produce a carbanion at C2, or homolytically to produce a
radical would result in highly unstable intermediates. Indeed,
we are unaware of a similar reaction in either the biological
realm or in conventional chemistry. Further studies are
planned to uncover the mechanism of this unique reaction.
The extremely low activity so far observed for the enzyme
presents a major barrier to its use in biofuels applications. Our
studies have identified conditions where the enzyme is much
more active, although substrate solubility appears to be an
important factor limiting catalytic rates. Moreover, an
enzyme that is capable of working in anoxic environments
may have practical advantages in technological applications.
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Received: March 2, 2011
Revised: May 17, 2011
Published online: June 10, 2011
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Keywords: biofuels · free radicals · hydrolysis reactions ·
.
metalloproteins · radical traps
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