[FeFe]-Hydrogenase Cyanide Ligands Derived from S-Adenosylmethionine- Dependent Cleavage of Tyrosine

Article abstract of DOI:10.1002/anie.200907047

"Chemical Equation Presented" What's your poison? Hydrogenases catalyze the reversible formation of dihydrogen from two electrons and two protons. The maturation of the [FeFe]-hydrogenase active-site cofactor (H cluster) requires three gene products, HydE

Full text of DOI:10.1002/anie.200907047

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Chemie
DOI: 10.1002/anie.200907047
Cyanide Biosynthesis
[FeFe]-Hydrogenase Cyanide Ligands Derived From
S-Adenosylmethionine-Dependent Cleavage of Tyrosine**
Rebecca C. Driesener, Martin R. Challand, Shawn E. McGlynn, Eric M. Shepard, Eric S. Boyd,
Joan B. Broderick, John W. Peters, and Peter L. Roach*
Hydrogenases catalyze the reversible reduction of protons to
yield molecular hydrogen (H2) and occur in three evolutio-
narily unrelated forms termed the [Fe]-, [FeFe]-, and [NiFe]-
hydrogenases.[1,2] [FeFe]-hydrogenases all contain a complex
active-site cofactor termed the H cluster (1, Scheme 1) that
consists of a regular [4Fe4S] cluster bridged by a shared
cysteine thiolate sulfur atom to a 2Fe subcluster with
biologically unique carbon monoxide, cyanide, and dithiolate
ligands.[3,4] The H cluster is biosynthesized in a stepwise
process in which generalized host cell machinery[5] is directed
towards the synthesis of a [4Fe4S] subcluster with subsequent
synthesis and insertion of the 2Fe subcluster by specialized
hyd encoded proteins, HydE, HydF, and HydG.[1,6] HydE and
HydG are radical S-adenosylmethionine (AdoMet) enzymes
thought to be responsible for the synthesis and proper
incorporation of the nonprotein ligands.[1,7–9] HydF has been
proposed to function as a scaffold for assembly of the
H cluster 2Fe subcluster and also to mediate its subsequent
insertion into HydA.[6,10] HydG has recently been shown to
catalyze radical-mediated tyrosine cleavage and generate
p-cresol,[11] which is a known fermentation product of several
anaerobes.[12,13] The HydG-catalyzed reaction is proposed to
be similar to that catalyzed by the thiamine biosynthetic
enzyme ThiH and to yield dehydroglycine as an intermedi-
ate.[14–16] It was hypothesized by Pilet et al.[11] that two
molecules of dehydroglycine condense at an FeS cluster on
HydG and result in generation of the dithiolate ligand.
Herein, we demonstrate that cyanide is a product of HydG-
catalyzed tyrosine cleavage, a result which clarifies the role of
HydG and indicates that tyrosine is the source of the cyanide
ligands in the H cluster.
To investigate the reaction products formed by HydG,
enzyme activity assays containing chemically reconstituted
HydG (on average 5.1 ꢀ 0.5 Fe per HydG), tyrosine, AdoMet,
and sodium dithionite were prepared. At selected time points,
assays were stopped by acidification, and the precipitated
protein was removed by centrifugation. HPLC-based analysis
methods were then used to measure the concentration of
reaction products in the supernatant, thus confirming the
turnover of AdoMet to yield deoxyadenosine (DOA), whilst
tyrosine was cleaved to yield p-cresol.[11]
Cyanide was detected and quantified after derivatization
by a modification of the method of Tracqui et al.[17] in which
the cyanide anion reacts with naphthalene-2,3-dicarbalde-
hyde (NDA) 5 and a primary amine (either taurine (6) or
N1,N1-dimethylethane-1,2-diamine (7)), thus generating the
fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives 8 and 9
(Figure 1A). After derivatization, HPLC analysis showed a
fluorescent peak which coeluted with a standard of the CBI
derivative (Figure 1B). The components required for cya-
nide-forming activity were tyrosine, AdoMet, a reducing
agent, and HydG. Omission of any of these components
resulted in loss of activity, consistent with a HydG-mediated
cleavage of tyrosine to form cyanide using radical AdoMet
chemistry. In these experiments, 5’-methylthioadenosine/
Scheme 1. Proposed biosynthesis of the HydA H cluster. HydA con-
tains a preformed [4Fe4S] cluster; HydE, HydG, and HydF then
function to form the 2Fe subcluster and non-protein-derived ligands
on HydF, which is then transferred to HydA. Recent spectroscopic
results suggest that X is an amine.[4]
[*] R. C. Driesener, M. R. Challand, Dr. P. L. Roach
School of Chemistry, University of Southampton
Southampton SO17 1BJ (UK)
Fax: (+44)2380-596-805
E-mail: plr2@soton.ac.uk
Homepage: http://www.soton.ac.uk/~roachweb/home/
index2.php
S. E. McGlynn, Dr. E. M. Shepard, Dr. E. S. Boyd, Prof. J. B. Broderick,
Prof. J. W. Peters
Department of Chemistry
and
Biochemistry and Astrobiology Biogeocatalysis Research Center
Montana State University, Bozeman, MT 59717 (USA)
[**] This work was supported by the Biotechnology and Biological
Sciences Research Council (M.R.C.), the Biochemical Society
(R.C.D.), the NASA Astrobiology Institute (J.B.B. & J.W.P.) and the
Airforce Office of Scientific Research (J.W.P.). S.E.M. is supported by
an NSF IGERT Fellowship (DGE 0654336). E.S.B. was supported by
NAI Postdoctoral Fellowship.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200907047.
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approximately 10 mol% per
mole glyoxylate was detected in
the solution. In optimized activ-
ity assays, the concentration of
glyoxylate was below 10% of the
HydG concentration, consistent
with it being an intermediate.
The stoichiometry of product
formation in HydG reactions was
investigated over
a one-hour
time course. Activity assays
were initiated by the addition of
dithionite and stopped at
selected time points (between 1
and 60 min) by acidification to
precipitate HydG and release
any protein-bound products into
solution. HPLC analysis with
UV/Vis detection allowed the
measurement of the products
DOA and p-cresol, whereas cya-
nide was detected as the CBI
fluorescent derivative 8. Accu-
rate measurement of cyanide
required careful calibration
using standard cyanide solutions
that were subjected to conditions
identical to the reaction and
workup conditions of HydG
activity assays. The time-course
data was fitted to a first-order
process (Figure 2 and Table 1)
and demonstrated that cyanide
and p-cresol were formed in a 1:1
ratio, with each HydG producing
Figure 1. Characterization of cyanide as a product of HydG catalyzed tyrosine cleavage. A) Formation of
cyanide from tyrosine by HydG and subsequent precolumn derivatization with amine 6 for fluorescent
HPLC or amine 7 for LC-MS analysis. B) Fluorescent HPLC analysis of CBI 8 from a negative control
HydG assay lacking tyrosine (g), the full assay (c), and a 50 mm synthetic standard of cyanide
(b). C) Mass spectrum of CBI derivative 9 prepared from an unlabeled cyanide standard. D) Mass
spectrum of CBI derivative 9 from an assay mixture incubated with l-U-[13C,15N]tyrosine.
S-adenosylhomocysteine nucleosidase (MTAN) was added to
the reaction mixture to hydrolyze the DOA formed in the
reaction, thus minimizing potential product inhibition[18] and
maximizing the formation of tyrosine-derived reaction prod-
ucts. Under these conditions, tyrosine cleavage reached 43%,
forming 430 mm p-cresol (Figure S2 in the Supporting Infor-
mation). To confirm that tyrosine is the source of cyanide,
l-U-[13C,15N]tyrosine was incubated with HydG, AdoMet,
and dithionite. After removal of HydG, the assay supernatant
was derivatized with NDA 5 and amine 7 and analyzed by LC-
MS. Relative to the product obtained with an unlabeled
cyanide standard, the product formed from labeled tyrosine
!
*
*
Figure 2. Time course of DOA ( ), p-cresol ( ), and cyanide ( )
formation from a HydG assay containing HydG (62 mm), tyrosine
(1 mm), AdoMet (1 mm), and sodium dithionite (1 mm).
resulted in a mass change of + 2 Da, consistent with the
formation of 13C,15N-labeled cyanide (Figure 2C,D).
ThiH and HydG have identical substrates and at least
three products in common (methionine, DOA, and p-cresol).
As the fourth reaction product of ThiH is dehydroglycine (10,
Scheme 2), we attempted to obtain evidence for the presence
of dehydroglycine associated with HydG. Dehydroglycine is
hydrolytically sensitive and has been detected during in vitro
activity assays by the formation of the hydrolysis product,
glyoxylate.[15] Purified HydG protein was denatured, and
4.7 mole equivalents of cyanide. The initial turnover number
for HydG (Table 1, kcat.0 = (20 ꢀ 2) ꢀ 10ꢁ4 sꢁ1) is comparable to
the observed activity of the closely related ThiH (for p-cresol
formation, kcat.0 = (32 ꢀ 8) ꢀ 10ꢁ4 sꢁ1).[16] The ratio of DOA to
p-cresol and cyanide was observed to be 1.3:1, indicating
some uncoupled turnover.[19] However, activity assays that
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enzyme aminocyclopropanecarboxylic acid oxidase (ACC
oxidase).[21] In bacteria, it has been shown that for the [NiFe]-
hydrogenase, cyanide ligands are generated from carbamoyl
phosphate through the initial formation of thiocyanate.[22]
This mechanism does not involve free cyanide as an
intermediate in the formation of the [NiFe]-hydrogenase
cofactor. For [FeFe]-hydrogenases, it is unclear whether
cyanide is released by HydG into free solution or is directly
transferred to another protein such as HydF. Several Pseu-
domonas species express a hydrogen cyanide synthase,[23]
which is proposed to oxidize glycine to generate dehydrogly-
cine as an intermediate[24] that upon oxidative decarboxyla-
tion yields cyanide.[25] Several potential mechanisms can
account for the formation of cyanide from tyrosine by
HydG. If the reaction proceeds via the postulated intermedi-
ate dehydroglycine (10), oxidative decarboxylation would
yield cyanide and carbon dioxide, but a decarbonylation
mechanism, which has chemical precedent,[26] would yield
both the cyanide and carbon monoxide ligands in a single step
(NHCHCO2H!HCN + CO + H2O).
ꢁ
Scheme 2. The Ca Cb lyase subfamily with currently characterized
biochemical functions.
In conclusion, we have demonstrated that HydG catalyzes
radical AdoMet chemistry, cleaving tyrosine to form a 1:1
stoichiometric ratio of cyanide to p-cresol. A slight excess of
DOA is generated, thus implying that a degree of uncoupled
AdoMet turnover occurs. The tyrosine-derived cyanide is
proposed to become associated with HydF, which functions as
a scaffold for H-cluster assembly, and the cluster is finally
transferred to HydA. Furthermore, this study provides the
first experimental evidence that some hydrogenase ligands
can be derived from the radical-mediated decomposition of
an amino acid and may have interesting implications in the
context of prebiotic chemistry.[27] The groundwork has now
been laid for further work focused on approaches to inves-
tigate the unresolved issues of the source of the dithiolate and
carbon monoxide ligands.
Table 1: Kinetic parameters of product formation.[a]
Product [Product]max [mm] k [ꢀ10ꢁ4 sꢁ1 [b]
]
kcat. [ꢀ10ꢁ4 sꢁ1 [b]
]
R2
0
DOA
373ꢀ11
5.2ꢀ0.3
5.2ꢀ0.5
4.3ꢀ0.4
31ꢀ3
24ꢀ3
20ꢀ2
0.99
0.99
0.99
p-cresol 281ꢀ15
cyanide 280ꢀ16
[a] Data from Figure 2 were fitted to a first-order process to determine
the final concentration of each product, the first-order rate constant, and
the initial turnover number. Values are shown with standard errors, and
R2 is a measure of the goodness of fit. [b] Rate constants (k) and turnover
numbers (kcat.0) were derived as described previously.[16]
contained AdoMet and the reductant but lacked tyrosine
showed very little uncoupled turnover (Figure S3 in the
Supporting Information), implying that, like ThiH, uncoupled
turnover likely proceeds by futile cycling involving a tyrosinyl
radical.[16]
Received: December 14, 2009
Published online: January 27, 2010
Sequence analysis of several radical AdoMet proteins
showed that HydG forms a small subfamily with two other
proteins, ThiH and NosL (Figure S5 in the Supporting
Information). NosL is a radical AdoMet enzyme required
for the rearrangement of tryptophan (11) to provide the
3-methylindolyl moiety 12 during the biosynthesis of the
Keywords: bioinorganic chemistry · biosynthesis · cyanides ·
hydrogenases · metalloenzymes
.
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