Enzyme Mechanisms
Very Important Paper
Structural Characterization of Two CO Molecules Bound to the
Nitrogenase Active Site
Trixia M. Buscagan
+
, Kathryn A. Perez
+
, Ailiena O. Maggiolo, Douglas C. Rees,* and
Thomas Spatzal*
Abstract:
As an approach towards unraveling the nitrogenase
mechanism, we have studied the binding of CO to the active-
site FeMo-cofactor. CO is not only an inhibitor of nitrogenase,
but it is also a substrate, undergoing reduction to hydrocarbons
(Fischer–Tropsch-type chemistry). The C
C bond forming
capabilities of nitrogenase suggest that multiple CO or CO-
derived ligands bind to the active site. Herein, we report
a crystal structure with two CO ligands coordinated to the
FeMo-cofactor of the molybdenum nitrogenase at 1.33
resolution. In addition to the previously observed bridging
CO ligand between Fe2 and Fe6 of the FeMo-cofactor, a new
ligand binding mode is revealed through a second CO ligand
coordinated terminally to Fe6. While the relevance of this state
to nitrogenase-catalyzed reactions remains to be established, it
highlights the privileged roles for Fe2 and Fe6 in ligand
binding, with multiple coordination modes available depend-
ing on the ligand and reaction conditions.
B
iological nitrogen (N
2
) fixation is catalyzed by nitrogenases
(N
2
ases). The most well-studied N
2
ase (molybdenum [Mo]
N
2
ase) consists of two component proteins: the Fe protein,
a homodimer which contains a [4Fe4S] cluster, and the MoFe
protein, a heterotetramer which contains two unique complex
metalloclusters per heterodimer.
[1–5]
During catalysis, the two
component proteins form a complex, promoting ATP-depen-
dent electron transfer from the Fe protein to the MoFe
protein.
[3]
Substrate reduction ultimately occurs at the multi-
metallic active site of the MoFe protein, called the FeMo-
cofactor, by a mechanism that remains enigmatic.
[6]
The
FeMo-cofactor is comprised of iron and molybdenum atoms
with an overall composition of [7Fe:9S:1C:1Mo]–
R
-homocit-
rate.
[7]
Alternative N
2
ases, featuring cofactors with V or Fe in
place of the Mo ion, are expressed under Mo-deficient
conditions.
[4,8,9]
The as-isolated state of the Mo N
2
ase active site does not
bind substrates, implying that the active site must be activated
for substrate binding.
[6,10]
Indeed, it has long been proposed
that the N
2
ase active site features multiple substrate binding
sites and that the formation of these binding sites requires the
particular substrate under turnover conditions; that is, the
cofactor is dynamic during catalysis.
[11,12]
Given the complex-
ity of the N
2
ase active site and the lack of ligand binding to the
as-isolated [7Fe:9S:1C:1Mo]–
R
-homocitrate cofactor form,
the nature of substrate (or inhibitor) coordination remained
elusive until defined in detail by high resolution structural
studies of ligand-bound Mo and vanadium [V] N
2
ases.
[2,10,13–16]
Evidence for the dynamic behavior of the cofactor was
provided by the observation that selenium could be sub-
stituted into a specialized group of sulfurs in the FeMo-
cofactor known as the belt sulfides, and could migrate through
these positions under turnover conditions.
[13]
Given the ability of N
2
ases to catalyze CO reduction to
hydrocarbons (Fischer–Tropsch-type chemistry),
[17–19]
it is
important to structurally characterize various CO binding
modes at the active site since this information could help
illuminate the mechanism of hydrocarbon formation at an
atomic level. In particular, for Mo N
2
ase, methane (CH
4
) was
not detected as a product of CO reduction; rather, higher
order hydrocarbons were detected, suggesting that multiple
CO-derived molecules could bind to the FeMo-cofactor at
a time.
[17]
We reported the initial crystal structure of a ligand
bound form of Mo N
2
ase from
Azotobacter vinelandii
(Av) in
which one of the belt sulfides, S2B, of the cofactor is displaced
by a carbon monoxide (CO) molecule (
Av1-CO
);
[10]
a similar
binding mode was subsequently demonstrated for the Av
vanadium nitrogenase.
[16]
Spectroscopic studies have high-
lighted that several distinct CO-bound species can be
observed under turnover conditions.
[20–23]
As the CO-binding
site(s), the nature of CO-binding, and the possibility of S2B
displacement could not be unequivocally established from the
spectroscopic data,
[24,25]
it became of interest to determine
how multiple CO molecules might bind to the FeMo-cofactor.
In this study we extend our previously established procedure
to structurally characterize ligand-bound and sulfide-substi-
tuted states of the FeMo-cofactor with an approach that
resulted in two CO molecules trapped at the active site.
[*] Dr. T. M. Buscagan,
[+]
Dr. K. A. Perez,
[+]
A. O. Maggiolo,
Prof. D. C. Rees, Dr. T. Spatzal
Division of Chemistry and Chemical Engineering
California Institute of Technology
1200 E. California Blvd., Pasadena, CA 91125 (USA)
E-mail: dcrees@caltech.edu
spatzal@caltech.edu
Dr. T. M. Buscagan,
[+]
Prof. D. C. Rees
Howard Hughes Medical Institute, California Institute of Technology
1200 E. California Blvd., Pasadena, CA 91125 (USA)
Dr. K. A. Perez
[+]
Present address: European Molecular Biology Laboratory
Meyerhofstrasse 1, 69117 Heidelberg (Germany)
[
+
] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202015751.
2020 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
A
ngewandte
Chemi
e
Communications
How to cite:
Angew. Chem. Int. Ed.
2021
,
60
, 5704–5707
International Edition: doi.org/10.1002/anie.202015751
German Edition:
doi.org/10.1002/ange.202015751
5704
2020
The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed.
2021
,
60
, 5704 –5707
The crystal structure solved at 1.33 resolution of a new
CO-bound state of the MoFe protein,
Av1(CO)
2
, is shown in
Figure 1.
Av1(CO)
2
was challenging to prepare due to the
weaker binding of the second CO and required pressurization
of
Av1-CO
crystals to 80 psi to maximize occupancy of that
site. It is important to note that
Av1(CO)
2
can only be
observed when
Av1-CO
is used as the starting material; that
is, FeMo-cofactor in the as-isolated Av1 protein is incapable
of binding CO on its own, consistent with previously reported
studies.
[1,10]
In the
Av1(CO)
2
structure, one CO ligand is
bridged between Fe2 and Fe6 in an analogous fashion to the
previously reported
Av1-CO
structure while a second CO
ligand is terminally bound to Fe6 (Figure 1A). The proximity
of the two CO ligands is intriguing, especially in the context of
C
C coupling at the Mo N
2
ase active site. Indeed, mechanistic
proposals for C
2
+
hydrocarbon formation include mono- or
bimetallic reductive elimination of CO-derived alkyl ligands
at one metal center or between neighboring metal centers.
Alternatively, migratory insertion mechanisms between CO
and CO-derived alkyl ligands are feasible.
[26]
Our structural
data suggest that these mechanistic proposals are possible as
Fe6 can accommodate two CO ligands and may serve as
either a single site for C
C bond formation or in a dinuclear
site in cooperation with Fe2.
In this structure, Fe6 adopts a five-coordinate trigonal
bipyramidal geometry as opposed to the four-coordinate
tetrahedral geometry observed for the other Fe centers in this
structure (including Fe2), as well as in the structures of the as-
isolated or
Av1-CO
crystal structures.
[7,10]
The terminal CO
oxygen atom interacts with the side chain amide N of
a
-
Gln191 (ca. 3.2 ), which may help stabilize substrate binding
at Fe6. Mutagenesis studies of
a
-Gln191 suggests the identity
of this residue affects ligand coordination and product
speciation in CO reduction reactions.
[18]
Additionally, the
terminal CO ligand is near the homocitrate moiety, and
previous work has shown that substitutions of the homocitrate
with structurally similar analogues decrease the substrate
reduction activity.
[27,28]
While the bridging CO ligand exhibits 100% occupancy at
both cofactors in the heterotetramer, the terminal CO moiety
exhibits approximately 50% occupancy, consistent with an
expected weaker association of the second CO molecule
under the experimental conditions. The presence of the
terminal CO was further validated through inspection of
polder omit maps calculated for this ligand (see SI, Fig-
ure S1).
[29]
In
Av1(CO)
2
, the terminal CO ligands exhibit
higher B-factors relative to the bridging COs, (see SI,
Table S2 and Figure S2). One explanation for this observation
involves a more dynamic association of the terminal CO,
which would result in greater positional displacements and
higher B-factors. A second explanation for the higher B-
factors is that the occupancy could be less than what is
currently modeled. Indeed, the reduced occupancy of the
terminal CO likely reflects partial dissociation caused by the
depressurization that necessarily occurs during cryo-protec-
tion of the crystal in preparation for X-ray diffraction data
collection. Because occupancies and B-factors are correlated,
we cannot distinguish between these two models.
[30]
With the
exception of Fe6, the distances between the interstitial carbon
and the Fe in the surrounding trigonal prism average (1.99
0.02) (Figure 1B), close to that observed for the as-isolated
3U7Q and
Av1-CO
structures ((2.00
0.01) and (1.99
0.02) , respectively). The Fe6-interstital carbon distance
increases by approximately 0.06 on the binding of the
second CO ligand, which is likely an underestimate due to the
fractional occupancy of the terminal CO ligand. Although the
increase of approximately 0.06 is comparable to the
estimated coordinate uncertainties (0.040 and 0.055 , for
Figure 1.
The FeMo-cofactor with two bound CO ligands [
Av1(CO)
2
]. Refined structure of
Av1(CO)
2
in the vicinity of the FeMo-cofactor at
a resolution of 1.33 . a) Side-view of the FeMo-cofactor highlighting the two CO ligands and the protein environment near the CO ligands.
b) Magnified view of the FeMo-cofactor in chain A with overlaid electron density (2
F
obs
F
calc
) map surrounding Fe2, Fe6, and CO atoms contoured
at 1.0
s
(represented as a blue mesh). Selected bond distances are shown. Iron atoms are shown in orange, sulfur in yellow, molybdenum in
turquoise, carbon in gray, nitrogen in blue, and oxygen in red.
A
ngewandte
Chemi
e
Communications
5705
Angew. Chem. Int. Ed.
2021
,
60
, 5704 –5707
2020
The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org
Av1(CO)
2
and
Av1-CO
, respectively),
[31,32]
it is consistent with
observations from synthetic studies in which changes to the Fe
geometry are enabled by a flexible Fe–X interaction, thus
allowing the Fe center to stabilize
p
-basic and
p
-acidic species
trans to ligand X.
[33–36]
To complement our structural data, we obtained EPR
spectroscopic data of the
Av1(CO)
2
pre- and postcrystalliza-
tion (solution and crystal slurry, respectively; Figure 2 and
SI). We observed that the solution behavior was consistent
with previously reported EPR studies with both hi- and lo-CO
species detected (with
g
=
2.17, 2.06 and
g
=
2.10, 1.98, and
1.92, respectively)
[20,24,37,38]
depending on whether an excess of
CO was present upon freezing the protein for storage and
during EPR sample preparation. Finally, and in line with our
crystallographic data, the EPR spectrum of a crystal slurry
consisting of
Av1(CO)
2
crystals confirms the presence of hi-
CO. The observation of the hi-CO EPR signal derived from
the addition of CO to crystallized
Av1-CO
is consistent with
previous reports in which lo-CO can be converted to hi-CO in
the absence of turnover conditions.
[21]
The Fischer–Tropsch-type chemistry exhibited by nitro-
genase suggests that the multimetallic active site can bind
more than one ligand simultaneously. The expansion of the
Fe6 coordination environment to accommodate a second CO
ligand represents a new mode of FeMo-cofactor ligand
binding that complements the displacement of belt sulfurs
by ligands that has been previously reported for Mo and V
N
2
ases.
[10,13,15,16,39]
For CO reduction to hydrocarbons, one
might intuit that binding of coupled substrates would occur at
one metal center or adjacent metal centers, leading to
reductive elimination of the hydrocarbon product. At least
for the first CO binding event at the cofactor, it has been
proposed that CO binds to the more oxidized face of the as-
isolated state of the cluster (as determined by spatially
resolved anomalous dispersion on the as-isolated state of the
MoFe protein),
[40]
which presumably becomes reduced under
turnover conditions. On the other hand, binding of the second
CO ligand does not require a change to the total oxidation
state of the FeMo-cofactor (although there could be internal
redox changes).
[41]
The binding of a terminal CO ligand to Fe6
is consistent with mutagenesis and spectroscopic studies
implicating Fe6 as a site for substrate binding.
[18,25]
We
speculate that hydrogen bonding interactions with Gln191
may promote CO binding at Fe6.
Previously reported
13/12
CO labeling studies by the Ribbe
and Hu groups suggest that the hi-CO form of V nitrogenase
is not a competent intermediate in CO coupling, while the lo-
CO form is catalytically competent.
[42]
Based on these results
and others,
[43,44]
a mechanistic hypothesis regarding CO
reduction has been proposed in which the first CO ligand is
reduced (at least partially) before a second CO molecule can
bind to the cofactor and undergo productive reduction.
[4,45,46]
While Vand Mo N
2
ase are structurally similar, they do exhibit
disparate activities towards CO reduction with the former
being much more reactive towards hydrocarbon formation.
Given these differences, it is entirely possible that the two
nitrogenases follow distinct mechanistic paths for CO reduc-
tion.
The FeMo-cofactor with two bound CO ligands may
provide a snapshot of how nature arranges CO-derived
ligands for Fischer–Tropsch-type chemistry. In particular,
Fe2 and Fe6 seem to be preferential sites for binding, with
various coordination modes available depending on the
identity of the incoming ligand. It is also plausible that
similar considerations are relevant for the mechanism of
dinitrogen reduction by nitrogenase. Dinitrogen is known to
coordinate to mono-, bi- and multimetallic sites via various
coordination modes, but only a handful of these dinitrogen
complexes lead to productive N
2
reduction products.
[6,47]
Determining whether certain substrate binding modes are
more prone to productive reduction pathways is of particular
interest in synthetic inorganic chemistry and now the active
site of N
2
ase faces similar questions.
Acknowledgements
We thank the Gordon and Betty Moore Foundation and the
Beckman Institute at Caltech for their generous support of
the Molecular Observatory at Caltech. We thank Prof.
James B. Howard, Dr. Rebeccah Warmack, Dr. Renee
Arias, Dr. Belinda Wenke, Dr. Stephanie Threatt, and
Siobhn MacArdle for insightful discussions, Dr. Jens
Kaiser for support of crystallographic data collection, Jeffrey
Lai for growing
Azotobacter vinelandii
, and Dr. Paul Oyala
for EPR training and support. Use of the Stanford Synchro-
tron Radiation Lightsource, SLAC National Accelerator
Laboratory, is supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences under
Contract No. DE-AC02-76SF00515. The SSRL Structural
Molecular Biology Program is supported by the DOE Office
of Biological and Environmental Research, and by the
National Institutes of Health, National Institute of General
Medical Sciences (including P41GM103393). This research
was supported by the National Institute of Health (NIH
Grant GM45162) and the Howard Hughes Medical Institute.
The Caltech EPR Facility is supported by NSF-1531940.
Figure 2.
EPR spectrum of a crystal slurry containing
Av1(CO)
2
. Exper-
imental data (black) and simulation (blue). For the full spectrum and
simulation parameters, see the Supporting Information. *Indicates the
signal diagnostic for previously reported hi-CO species.
A
ngewandte
Chemi
e
Communications
5706
www.angewandte.org
2020
The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed.
2021
,
60
, 5704 –5707
Conflict of interest
The authors declare no conflict of interest.
Keywords:
carbonyl ligands · C
C coupling · cofactors ·
nitrogenases · X-ray diffraction
[1] B. K. Burgess, D. J. Lowe,
Chem. Rev.
1996
,
96
, 2983 – 3012.
[2] O. Einsle, D. C. Rees,
Chem. Rev.
2020
,
120
, 4969 – 5004.
[3] H. L. Rutledge, F. A. Tezcan,
Chem. Rev.
2020
,
120
, 5158 – 5193.
[4] A. J. Jasniewski, C. C. Lee, M. W. Ribbe, Y. Hu,
Chem. Rev.
2020
,
120
, 5107 – 5157.
[5] L. C. Seefeldt, Z.-Y. Yang, D. A. Lukoyanov, D. F. Harris, D. R.
Dean, S. Raugei, B. M. Hoffman,
Chem. Rev.
2020
,
120
, 5082 –
5106.
[6] T. M. Buscagan, D. C. Rees,
Joule
2019
,
3
, 2662 – 2678.
[7] T. Spatzal, M. Aksoyoglu, L. Zhang, S. L. A. Andrade, E.
Schleicher, S. Weber, D. C. Rees, O. Einsle,
Science
2011
,
334
,
940.
[8] R. R. Eady,
Chem. Rev.
1996
,
96
, 3013 – 3030.
[9] N. S. Sickerman, Y. Hu, M. W. Ribbe,
Methods Mol. Biol.
2019
,
1876
, 3 – 24.
[10] T. Spatzal, K. A. Perez, O. Einsle, J. B. Howard, D. C. Rees,
Science
2014
,
345
, 1620 – 1623.
[11] J. C. Hwang, C. H. Chen, R. H. Burris,
Biochim. Biophys. Acta
1973
,
292
, 256 – 270.
[12] J. M. Rivera-Ortiz, R. H. Burris,
J. Bacteriol.
1975
,
123
, 537 – 545.
[13] T. Spatzal, K. A. Perez, J. B. Howard, D. C. Rees,
eLife
2015
,
4
,
11620 – 11630.
[14] D. Sippel, O. Einsle,
Nat. Chem. Biol.
2017
,
13
, 956 – 961.
[15] D. Sippel, M. Rohde, J. Netzer, C. Trncik, J. Gies, K. Grunau, I.
Djurdjevic, L. Decamps, S. L. Andrade, O. Einsle,
Science
2018
,
359
, 1484 – 1489.
[16] M. Rohde, K. Grunau, O. Einsle,
Angew. Chem. Int. Ed.
2020
,
59
,
23626 – 23630.
[17] Y. Hu, C. C. Lee, M. W. Ribbe,
Science
2011
,
333
, 753.
[18] Z.-Y. Yang, D. R. Dean, L. C. Seefeldt,
J. Biol. Chem.
2011
,
286
,
19417 – 19421.
[19] L. C. Seefeldt, Z.-Y. Yang, S. Duval, D. R. Dean,
Biochim.
Biophys. Acta
2013
,
1827
, 1102 – 1111.
[20] L. C. Davis, M. T. Henzl, R. H. Burris, W. H. Orme-Johnson,
Biochemistry
1979
,
18
, 4860 – 4869.
[21] R. C. Pollock, H.-I. Lee, L. M. Cameron, V. J. DeRose, B. J.
Hales, W. H. Orme-Johnson, B. M. Hoffman,
J. Am. Chem. Soc.
1995
,
117
, 8686 – 8687.
[22] L. Yan, C. H. Dapper, S. J. George, H. Wang, D. Mitra, W. Dong,
W. E. Newton, S. P. Cramer,
Eur. J. Inorg. Chem.
2011
, 2064 –
2074.
[23] L. Yan, V. Pelmenschikov, C. H. Dapper, A. D. Scott, W. E.
Newton, S. P. Cramer,
Chem. Eur. J.
2012
,
18
, 16349 – 16357.
[24] H.-I. Lee, L. M. Cameron, B. J. Hales, B. M. Hoffman,
J. Am.
Chem. Soc.
1997
,
119
, 10121 – 10126.
[25] H.-I. Lee, M. Sørlie, J. Christiansen, T.-C. Yang, J. Shao, D. R.
Dean, B. J. Hales, B. M. Hoffman,
J. Am. Chem. Soc.
2005
,
127
,
15880 – 15890.
[26] J. F. Hartwig,
Organotransition Metal Chemistry: From Bonding
to Catalysis
, University Science Books, Sausalito,
2010
.
[27] J. Imperial, T. R. Hoover, M. S. Madden, P. W. Ludden, V. K.
Shah,
Biochemistry
1989
,
28
, 7796 – 7799.
[28] S. M. Mayer, C. A. Gormal, B. E. Smith, D. M. Lawson,
J. Biol.
Chem.
2002
,
277
, 35263 – 35266.
[29] D. Liebschner, P. V. Afonine, N. W. Moriarty, B. K. Poon, O. V.
Sobolev, T. C. Terwilliger, P. D. Adams,
Acta Crystallogr. Sect. D
2017
,
73
, 148 – 157.
[30] B. Rupp,
Biomolecular Crystallography: Principles, Practice, and
Application to Structural Biology
, Garland Science, New York,
2009
.
[31] A. A. Vaguine, J. Richelle, S. J. Wodak,
Acta Crystallogr. Sect. D
1999
,
55
, 191 – 205.
[32] D. W. J. Cruickshank,
Acta Crystallogr. Sect. D
1999
,
55
, 583 –
601.
[33] Y. Lee, N. P. Mankad, J. C. Peters,
Nat. Chem.
2010
,
2
, 558 – 565.
[34] M.-E. Moret, J. C. Peters,
Angew. Chem. Int. Ed.
2011
,
50
, 2063 –
2067;
Angew. Chem.
2011
,
123
, 2111 – 2115.
[35] J. Rittle, J. C. Peters,
Proc. Natl. Acad. Sci. USA
2013
,
110
,
15898 – 15903.
[36] S. E. Creutz, J. C. Peters,
J. Am. Chem. Soc.
2014
,
136
, 1105 –
1115.
[37] P. D. Christie, H.-I. Lee, L. M. Cameron, B. J. Hales, W. H.
Orme-Johnson, B. M. Hoffman,
J. Am. Chem. Soc.
1996
,
118
,
8707 – 8709.
[38] H.-I. Lee, B. J. Hales, B. M. Hoffman,
J. Am. Chem. Soc.
1997
,
119
, 11395 – 11400.
[39] W. Kang, C. C. Lee, A. J. Jasniewski, M. W. Ribbe, Y. Hu,
Science
2020
,
368
, 1381 – 1385.
[40] T. Spatzal, J. Schlesier, E.-M. Burger, D. Sippel, L. Zhang,
S. L. A. Andrade, D. C. Rees, O. Einsle,
Nat. Commun.
2016
,
7
,
10902 – 10908.
[41] C. H. Arnett, M. J. Chalkley, T. Agapie,
J. Am. Chem. Soc.
2018
,
140
, 5569 – 5578.
[42] C. C. Lee, J. Wilcoxen, C. J. Hiller, R. D. Britt, Y. Hu,
Angew.
Chem. Int. Ed.
2018
,
57
, 3411 – 3414;
Angew. Chem.
2018
,
130
,
3469 – 3472.
[43] C. C. Lee, Y. Hu, M. W. Ribbe,
ACS Cent. Sci.
2018
,
4
, 1430 –
1435.
[44] C. J. Hiller, C. C. Lee, M. T. Stiebritz, L. A. Rettberg, Y. Hu,
Chem. Eur. J.
2019
,
25
, 2389 – 2395.
[45] I. Dance,
Dalton Trans.
2011
,
40
, 5516 – 5527.
[46] J. A. Buss, G. A. Bailey, J. Oppenheim, D. G. VanderVelde,
W. A. Goddard, T. Agapie,
J. Am. Chem. Soc.
2019
,
141
, 15664 –
15674.
[47] R. J. Burford, M. D. Fryzuk,
Nat. Rev. Chem.
2017
,
1
, 0026.
Manuscript received: November 25, 2020
Accepted manuscript online: December 15, 2020
Version of record online: January 27, 2021
A
ngewandte
Chemi
e
Communications
5707
Angew. Chem. Int. Ed.
2021
,
60
, 5704 –5707
2020
The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org