of 11
Ligand binding to the FeMo-cofactor: structures of CO-bound
and reactivated nitrogenase
Thomas Spatzal
1,*
,
Kathryn A. Perez
1
,
Oliver Einsle
2,3
,
James B. Howard
1,4
, and
Douglas C.
Rees
1,*
1
Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, Mail
Code 114-96, California Institute of Technology, Pasadena, CA 91125, USA.
2
Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany.
3
BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, 79104
Freiburg, Germany.
4
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota,
Minneapolis, MN 55455, USA.
Abstract
The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how
inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide
(CO) inhibited nitrogenase MoFe-protein at 1.50 Å resolution, revealing a CO molecule bridging
Fe2 and Fe6 of the FeMo-cofactor. The μ
2
binding geometry is achieved by replacing a belt-sulfur
atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement
of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and
reappearance of S2B in the 1.43 Å resolution structure of the reactivated enzyme. The substantial
and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated
and provides insights into a catalytically competent state of nitrogenase.
Biological nitrogen fixation is Nature‘s pathway to convert atmospheric dinitrogen (N
2
) into
a bioavailable form, ammonia (NH
3
). Nitrogenase, the only known enzyme capable of
performing this multi-electron reduction, consists of two component metalloproteins, the Fe-
(Av2) and MoFe- (Av1) protein (
1
3
). The Fe-protein, containing a [4Fe:4S]-cluster,
mediates the adenosine triphosphate (ATP) dependent electron transfer to the MoFe-protein
to support dinitrogen reduction (
4
). The MoFe-protein is an
α
2
β
2
heterotetramer with one
catalytic unit per
αβ
heterodimer (
5
). To achieve the elaborate redox properties required for
*
Correspondence to: TS, spatzal@caltech.edu, phone 1-626-395-2047, DCR, dcrees@caltech.edu, phone 1-626-395-8393, fax
1-626-744-9524, (address manuscript review correspondence to DCR).
“This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the
complete version of record at
http://www.sciencemag.org/
. The manuscript may not be reproduced or used in any manner that does not
fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS.”
Supplementary Materials:
Materials and Methods
Figures S1&2
Tables S1&2
References 36–44
NIH Public Access
Author Manuscript
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. Author manuscript; available in PMC 2015 September 26.
Published in final edited form as:
Science
. 2014 September 26; 345(6204): 1620–1623. doi:10.1126/science.1256679.
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reducing the N-N triple bond, two metal centers are present in the MoFe-protein: the P-
cluster and the FeMo-cofactor. The P-cluster, an [8Fe:7S] entity, is the initial acceptor for
electrons, donated from the Fe-protein during complex formation between the two proteins
(
6
8
). Electrons are subsequently transferred to the FeMo-cofactor, a [7Fe:9S:C:Mo]-
R
-
homocitrate cluster that constitutes the active site for substrate reduction, and is the most
complex metal center known in biological systems (
5
,
9
12
).
Substrates and inhibitors bind only to forms of the MoFe-protein reduced by two to four
electrons relative to the resting, "as-isolated" state, which can only be generated in the
presence of reduced Fe-protein and ATP (
1
). Mechanistic studies must take into account the
dynamic nature of the nitrogenase system, requiring multiple association and dissociation
events between the two component proteins, as well as the ubiquitous presence of protons
that are reduced to dihydrogen even in competition with other substrates (
1
,
13
15
). The
resulting distribution of intermediates under turnover conditions significantly complicates
the structural and spectroscopic investigation of substrate interactions. Hence, even the
fundamental question whether molybdenum or iron represents the site for substrate binding
at the FeMo-cofactor is still under debate, and as a consequence, a variety of mechanistic
pathways have been proposed based on either molybdenum or iron as the catalytic center
mainly following Chatt-type chemistry (
16
).
Inhibitors are potentially powerful tools for the preparation of stably trapped transient states
that could provide insight into the multi-electron reduction mechanism. In this regard,
carbon monoxide (CO), a non-competitive inhibitor for all substrates except protons (
17
,
18
), has a number of attractive properties; CO is isoelectronic to the physiological substrate,
is a reversible inhibitor, and only binds to partially reduced MoFe-protein generated under
turnover conditions. While non-competitive inhibitors are traditionally considered to bind at
distinct sites from the substrate, for complex enzymes such as nitrogenase with multiple
oxidation states and potential substrate binding modes, this distinction is not required (
19
).
More recently, it has also been shown that CO is a substrate, albeit a very poor one, whose
reduction includes concomitant C-C bond formation to generate C2 and longer-chain
hydrocarbons, in a reaction reminiscent of the Fischer-Tropsch synthesis (
20
,
21
). Therefore,
CO binding as inhibitor/substrate must involve important active site properties common to
the reduction of the natural substrate dinitrogen. For this reason, CO binding has been
investigated by a variety of spectroscopic methods, most notably EPR and IR, and
depending on the partial pressure, multiple CO-bound species have been observed; yet, a
structurally explicit description of any CO binding site has been elusive (
18
,
22
27
).
Building on these observations, we have determined a high-resolution crystal structure of a
CO-bound state of the MoFe-protein from
Azotobacter vinelandii
. This necessitated
overcoming several obstacles. First, the experimental setup for all protein handling steps,
including crystallization, was deemed to require the continuous presence of CO. Second,
because inhibition requires enzyme turnover, a prerequisite was the ability to obtain crystals
of the MoFe-protein from activity assay mixtures, rather than from isolated protein (see
supplementary material for assay details), conditions that are typically contradictory to
crystallization requirements. Finally, rapid MoFe-protein crystallization (≤ 5 hrs) was
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crucial and was achieved based on previously developed protocols in combination with
seeding strategies, while maintaining the presence of CO throughout this process (
10
).
Crystals of the inhibited MoFe-protein (Av1-CO) yielded structural data at 1.50 Å
resolution, which allowed clear identification of a CO ligand (Figure 1A, C, S1A–D). The
Av1-CO structure directly demonstrates the binding of one molecule CO per active site in a
μ
2
-bridging mode between Fe2 and Fe6 that form one edge of the trigonal six-iron prism
(Fe
2-3-4-5-
6-7) of the FeMo-cofactor (Fig. 1A, C, D). Remarkably, CO binding is
accompanied by a displacement of one of the belt sulfur atoms (S2B) while retaining the
essentially tetrahedral coordination spheres for Fe2 and Fe6. As a result, the two Fe are
coordinated by two sulfur and two carbon atoms, a geometry, to our knowledge, not
previously observed in metallo-clusters (although higher coordination number geometries
have been observed in FeFe-hydrogenases (
28
)). Confirmation of the S2B displacement was
provided by anomalous difference Fourier maps calculated with diffraction data measured at
7100 eV (Fig. 1B); this energy is just below the Fe K-edge so that the anomalous scattering
from S is significantly enhanced relative to Fe. The carbon atom of CO is located at a
distance of 1.86 Å from each of the irons (Fe2 and Fe6), compared to a previous distance of
2.2 Å for S2B (Figure 1C). The altered ligand environment results in a small adjustment of
the FeMo-cofactor geometry, with the Fe2–Fe6 distance (2.5 Å) slightly shortened relative
to the unchanged Fe4–Fe5 and Fe3–Fe7 distances (2.6 Å) (Figure 1C).
Given the complete displacement of S2B, we assessed whether the CO-inhibited protein
could be reactivated or if it was irreversibly modified. Crystals of CO-inhibited MoFe-
protein were active when dissolved in an assay mixture in the absence of CO. Furthermore,
when CO-inhibited MoFe-protein from the original inhibition preparations was newly
assayed after removal of CO (see supplementary material for assay details), a quantitative
recovery (94 ± 4 %) of the initial activity was obtained (Table 1). The reactivated MoFe-
protein was subsequently re-isolated from activity assay mixtures and crystallized, yielding
a structure at 1.43 Å resolution. The structural data of the protein (Av1-reactivated) revealed
that S2B is regained by replacing the previously bound CO-ligand, which results in the
recovery of the resting state FeMo-cofactor. The full occupancy of the sulfur at the S2B-site
in the reactivated enzyme is evident by inspecting the 2F
o
-F
c
electron density map as well as
the anomalous electron density map verifying the anomalous scattering contribution
expected for S2B (Figure 2A, B).
The finding that S2B can be reversibly replaced by CO raises the question of where this
atom is located in the CO-inhibited state. If the S2B binding site is ordered, candidate
locations should be evident by an inspection of the 7100 eV anomalous difference Fourier
map. In this manner, one site per catalytic unit was identified with anomalous density
compatible with sulfur. This potential sulfur binding site (SBS) is positioned ~22 Å away
from the S2B position in the FeMo-cofactor and consists of a small protein pocket at the
interface of the
α
- and
β
-subunits, formed by the side chains of residues
α
-Arg93,
α
-Thr104,
α
-Thr111,
α
-Met112,
β
-Asn65,
β
-Trp428,
β
-Phe450 and
β
-Arg453 (Figure 3A, B). The
positive surface charge of the cavity is suited to accommodate an anionic species such as
HS
or S
2−
(Figure 3B). In previous structures of the resting state enzyme (pdb-IDs: 1M1N
and 3U7Q), this site has been assigned as water; intriguingly, the density at this site is also
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decreased in the structure of reactivated Av1 (Figure S2). The potential SBS is connected to
the FeMo-cofactor binding pocket by a non-continuous water channel, and conformational
rearrangements would be needed to accommodate the reversible migration of S2B from and
to the active site. Although we have observed a correlation between density at the potential
SBS and the CO-inhibited state of the MoFe-protein, the identity of this site as the displaced
S2B cannot be achieved solely based on crystallographic data. In assessing the relevance of
this site, it should be noted that the residues forming the pocket are poorly conserved, the
site seems rather remote from the FeMo-cofactor, and since sulfur and chloride have similar
anomalous scattering properties at 7100 eV, it is possible that this is a general anion-binding
site in Av1.
The crystallographic characterization of CO-bound FeMo-cofactor of the MoFe-protein has
important implications for the mechanism of substrate reduction by nitrogenase:
The CO binding site is close to the side chains of residues
α
-His195 (2.8 Å, NE2-OC
distance) and
α
-Val70 (3.4 Å, closest methyl-OC distance). Modifications to both side
chains were reported to significantly alter the catalytic properties of the enzyme. An
α
-
His195 to
α
-Gln195 mutation resulted in the loss of N
2
reduction activity while an
α
-Val70
to
α
-Ala/Gly70 alteration was reported to enable the reduction of longer carbon-chain
substrates such as propyne and 1-butyne, respectively (
29
33
). In the structure presented
here,
α
-His195 is in hydrogen bonding distance to the oxygen of CO while
α
-Val70 directly
flanks the binding site (Figure 1D).
The displacement of S2B could be facilitated by a proton donation from
α
-His195 to yield
either HS
or H
2
S, thereby generating a better leaving group than S
2−
. Although the
dissociation of a sulfur may seem surprising, it opens up the ligand binding site, since the
large radius of S
2−
effectively shields the cofactor Fe atoms in the resting state from
substrate/inhibitor attack (
2
). The more general implication that binding of exogenous
ligands can be accompanied by the reversible dissociation of at least one belt-sulfur from the
metal sites of the FeMo-cofactor, changes the present view of the structural inertness of the
[7Fe:9S:C:Mo]-
R
-homocitrate cluster towards ligand exchange. The relative lack of
reactivity of the resting state is a striking property of the FeMo-cofactor, and the
requirement for more highly reduced forms to bind substrate and inhibitors may reflect the
need to dissociate sulfur ligands from Fe sites.
The displacement of the belt sulfur S2B by carbon monoxide causes the FeMo-cofactor
scaffold to lose its intrinsic three-fold symmetry. Additionally, Fe1, the interstitial carbon
and molybdenum are no longer aligned, creating an asymmetry in the resulting [7Fe:
8S:C:Mo] cluster (Figure 1C). The modest adjustments of the remaining scaffold upon CO
binding are suggestive of an important role for the interstitial carbon in stabilizing the
cofactor during rearrangements and substitutions to the coordination environment of the
irons (
34
,
35
).
The experimental manipulations used to generate the CO-inhibited structure are distinct
from those reported in previous spectroscopic studies; hence it is not possible to
unambiguously assign the structure to one of the many annotated spectroscopic states.
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Interestingly, many of the previously identified states undergo dynamic interchanges
including photoinduced transitions between states (
25
). Like the structure presented here, the
spectroscopically identified “lo-CO” state has been proposed to involve one molecule of CO
bound to the active site in a bridging mode (
22
,
26
). A state with two CO bound to Fe2 and
Fe6 could correspond to the “high-CO” form (
22
,
23
) and might represent an intermediate
relevant to the C-C coupling reaction.
The generation and successful stabilization of CO-bound MoFe protein under turnover
conditions has culminated in a crystal structure that provides a detailed view of a ligand
bound to the nitrogenase active site. The observations that CO is isoelectric to N
2
, is a potent
yet reversible inhibitor of substrate reduction without impeding proton reduction to
dihydrogen, and is bound in close proximity to previously determined catalytically
important residues emphasizes the relevance of the CO-bound structure towards
understanding dinitrogen binding and reduction. This sheds light on N
2
activation based on a
di-iron edge of the FeMo-cofactor and in this respect resembles the Haber-Bosch catalyst
that also uses an iron surface to break the N-N triple bond. The demonstrated structural
accessibility of CO-bound MoFe-protein opens the door for comparable studies on a variety
of inhibitors and substrates, with the goal of understanding the detailed molecular
mechanism of dinitrogen reduction by nitrogenase.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank J. Peters, L. Zhang, J. Rittle, C. Morrison, B. Wenke and K. Dörner for informative discussions. This
work was supported by NIH grant GM45162 (D.C.R.), Deutsche Forschungsgemeinschaft grants EI-520/7 and
RTG 1976 and the European Research Council N-ABLE project (O.E.). We gratefully acknowledge the Gordon
and Betty Moore Foundation, the Beckman Institute, and the Sanofi–Aventis Bioengineering Research Program at
Caltech for their generous support of the Molecular Observatory at Caltech, and the staff at Beamline 12–2,
Stanford Synchrotron Radiation Lightsource (SSRL) for their assistance with data collection. SSRL is operated for
the DOE and supported by its OBER and by the NIH, NIGMS (P41GM103393) and the NCRR (P41RR001209).
We thank the Center for Environmental Microbial Interactions for their support of microbiology research at
Caltech. Coordinates and structure factors have been deposited in the Protein Data Bank of the Research
Collaboratory for Structural Bioinformatics, with IDs 4TKV (Av1-CO) and 4TKU (Av1-reactivated).
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Figure 1. CO-inhibited MoFe-protein (Av1-CO)
Refined structure of the CO-bound FeMo-cofactor at a resolution of 1.50 Å. A) View along
the Fe1-C-Mo direction. The electron density (2F
o
-F
c
) map is contoured at 4.0
σ
and
represented as blue mesh. The density at the former S2B site is significantly decreased and
in excellent agreement with bound CO (see also C)). B) Same orientation as A)
superimposed with the anomalous density map calculated at 7100 eV (green) at a resolution
of 2.1 Å contoured at 4.0
σ
showing the significant reduction of anomalous electron density
at the CO site. C) Side view of FeMo-cofactor highlighting the μ
2
binding geometry of CO.
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The electron density (2F
o
-F
c
) map (blue mesh) surrounding CO-Fe2-Fe6-C is contoured at
1.5
σ
. D) Same orientation as C) highlighting the ligand environment of the metal center.
The catalytically important side chain residues
α
-Val70 and
α
-His195 are in close proximity
to the CO-binding site. Iron atoms are shown in orange, sulfur in yellow, molybdenum in
turquoise, carbon in grey, nitrogen in blue and oxygen in red.
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Figure 2. Reactivated MoFe-protein (Av1-reactivated)
Refined structure of the FeMo-cofactor at a resolution of 1.43 Å. A) View along the Fe1-C-
Mo direction. The electron density (2F
o
-F
c
) map is contoured at 4.0
σ
and represented as
blue mesh. Electron density at the S2B site is in excellent agreement with a regained sulfur.
B) Same orientation as A) superimposed with the anomalous density map (green) at a
resolution of 2.15 Å contoured at 4.0
σ
showing the presence of anomalous density at the
S2B site. Color scheme is according to Figure 1.
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Figure 3. Overview of the potential sulfur binding site (SBS) in the CO-inhibited MoFe-protein
(Av1-CO)
A) Location of the potentially bound sulfur in a protein cavity on the interface between the
α
- and
β
-subunit of the
α
2
β
2
MoFe-protein. The potential SBS is located 22 Å away from its
former position in the FeMo-cofactor (S2B-site). B) Close-up view on the binding cavity.
Positive surface charge is represented in blue, negative surface charge in red. The anomalous
density map at a resolution of 2.1 Å is represented as green mesh and contoured at 4.0
σ
showing the presence of anomalous density at the potential SBS. The side chain sulfur of
α
-
Met112 provides an internal standard for full occupancy. The color scheme is according to
Figure 1.
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Table 1
Nitrogenase activity
The acetylene reduction activity of “as-isolated” Av1, Av1-CO and Av1-reactivated was measured by the
quantification of ethylene production. Nitrogenase activity is quantitatively recovered upon reactivation.
Errors represent standard deviations of three measurements.
Sample
Specific reduction activity
[nmol(acetylene) min
−1
mg(Av1)
−1
]
Specific reduction activity
[%]
Av1-as-isolated
1930 ± 90
100 ± 5
Av1-CO
< 2 ± 2
< 0.1 ± 0.1
Av1-reactivated
1820 ± 80
94 ± 4
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