of 10
Article
https://doi.org/10.1038/s41467-023-36636-4
Structural consequences of turnover-
induced homocitrate loss in nitrogenase
Rebeccah A. Warmack
1
, Ailiena O. Maggiolo
1
, Andres Orta
2
,
Belinda B. Wenke
1
,JamesB.Howard
3
& Douglas C. Rees
1,4
Nitrogenase catalyzes the ATP-depende
ntreductionofdinitrogentoammonia
during the process of biological nitrogen
fi
xation that is essential for sus-
taining life. The active site FeMo-cof
actor contains a [7Fe:1Mo:9S:1C] metal-
locluster coordinated with an
R
-homocitrate (HCA) molecule. Here, we
establish through single particle cryoEM and chemical analysis of two forms of
the
Azotobacter vinelandii
MoFe-protein
a high pH turnover inactivated
species and a
Δ
NifV variant that cannot synthesize HCA
that loss of HCA is
coupled to
α
-subunit domain and FeMo-cofact
or disordering, and formation
of a histidine coordination site. We further
fi
nd a population of the
Δ
NifV
variant complexed to an en
dogenous protein identi
fi
ed through structural and
proteomic approaches as the uncharact
erized protein NafT. Recognition by
endogenous NafT demonstrates the p
hysiological relevance of the HCA-
compromised form, perhaps for cofactor
insertion or repair. Our results point
towards a dynamic active site in which HCA plays a role in enabling nitrogenase
catalysis by facilitating activation of the
FeMo-cofactor from a relatively stable
form to a state capable of reducing di
nitrogen under ambient conditions.
Biological nitrogen
fi
xation occurs within diazotrophic organisms
primarily via the Mo-nitrogenase
1
4
.Thisenzymeconsistsoftwoair-
sensitive metalloproteins, the catalytic molybdenum iron (MoFe-)
protein, and the reductase iron (Fe-) protein. The Fe-protein mediates
ATP-dependent electron transfer (ET) through its [4Fe:4S] cluster to
the intermediate [8Fe:7S] P-cluster in the MoFe-protein; the electrons
are subsequently shuttled to a [7Fe:1Mo:9S:1C]-
R
-homocitrate metal-
locluster, the FeMo-cofactor, within the active site of the MoFe-
protein. The reduction of dinitrogen to ammonia catalyzed by nitro-
genase proceeds at ambient temperatures and pressures, and is
increasingly of interest as an alternative to the industrial processes for
ammonia and hydrogen production, as well as for the reduction
of carbon-containing substrates to unsaturated hydrocarbons for
biofuels
5
,
6
. Nitrogenase further serves as a model system for the study
of biological metal-catalyzed enzymatic reactions. Despite its sig-
ni
fi
cance, the atomic basis for the mechanism is incomplete due to the
transient nature of its numerous intermediate states and the chal-
lenges of working with these oxygen-sensitive proteins
7
.
The FeMo-cofactor remains a primary focus of nitrogenase
research efforts, as substrates are expected to bind to and be reduced
by this metallocluster. This ligand is attached to the catalytic MoFe-
protein through two residues linked to Fe1 and Mo at opposing ends of
the cluster; the coordination sphere of Mo is completed by
R
-homo-
citrate (HCA) in a bidentate manner. HCA has been proposed to par-
ticipate in proton transfer to the active site
8
as well as to undergo
changes in Mo coordination that could open up sites for substrate
binding
9
11
. Structure-activity studies conducted by Ludden and cow-
orkers have established that the key features of the HCA ligand for
substrate reduction include a hydroxyl group and a minimum of two
carboxyl groups
12
. However, the precise structural and chemical role
played by HCA in nitrogenase nitrogen
fi
xation remains unclear. Given
the possible involvement of HCA in proton transfer, changes in pH
Received: 21 October 2022
Accepted: 9 February 2023
Check for updates
1
Division of Chemistry and Chemical Engineering 147-75, Ca
lifornia Institute of Technology, Pasadena, CA 91125, USA.
2
Biochemistry and Molecular Biophysics
Graduate Program, California Institute of Technology, Pasadena, CA 91125, USA.
3
Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455,
USA.
4
Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.
e-mail:
rwarmack@caltech.edu
;
dcrees@caltech.edu
Nature Communications
| (2023) 14:1091
1
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present an avenue for probing the role of this moiety in catalysis. A
mechanism-based inactivated state of nitrogenase prepared by turn-
over under high pH conditions has been described
13
. Yang et al. noted
that increased pH drove progressive, partially reversible inhibition of
the MoFe-protein in a
fi
rst-order process, and
fi
nally inactivation, with
increased inactivation rates observed at higher pHs. The structure of
that high pH turnover-inactivated state is still undetermined, as it has
proven recalcitrant to crystallization.
In this work, we sought to determine the structure of this
turnover-induced state, here referred to as the MoFe
Alkaline-inactivated
state,
by employing the cryo-electron microscopy (cryoEM) method of sin-
gle particle analysis. A resulting 2.37 Å resolution structure of
MoFe
Alkaline-inactivated
demonstrates that HCA is lost from the active site
under these conditions. We develop a biochemical assay for the
detection of HCA from protein samples, and con
fi
rm that intact HCA
cannot be detected in samples of MoFe
Alkaline-inactivated
.Inthisstructure,
the loss of HCA couples to an asymmetric
α
-subunit domain and FeMo-
cofactor disordering, and the formation of a histidine quartet near the
more disordered active site. Complementary structural and bio-
chemical analysis of MoFe-protein puri
fi
ed from a homocitrate syn-
thase (
Δ
nifV
)deletionstrainof
Azotobacter vinelandii
,termedthe
MoFe
Δ
NifV
state, reveals loss of HCA and similar structural features.
Intriguingly, reconstructions from a subset of particles of this puri
fi
ed
protein reveal MoFe
Δ
NifV
complexed to an endogenous protein that we
have identi
fi
ed through a combination of structural and proteomic
approaches, and the structural prediction program AlphaFold, as the
previously uncharacterized protein Avin_01560 (also annotated as
NafT) present in the
nif
operon region of the
A. vinelandii
genome
14
,
15
.
This complex demonstrates the physiological relevance of the
homocitrate-compromised form of the MoFe-protein, as NafT speci-
fi
cally recognizes this state. Our results point towards a dynamic active
site in which homocitrate plays an active role in enabling the chemistry
of nitrogenase catalysis as part of a transformation where the FeMo-
cofactor is activated from a relatively stable form to a state capable of
reducing dinitrogen under ambient conditions. This work demon-
strates that increasingly powerful cryoEM techniques can fundamen-
tally advance our understanding of complex metalloenzyme
mechanisms by revealing previously uncharacterized states of rele-
vance to turnover, assembly, or repair.
Results
Anaerobic cryoEM structures of MoFe-protein
Single particle cryoEM is ideally suited to interrogate this system given
its increasing gains in resolution
16
,
17
and ability to anaerobically probe
macromolecular structures
18
20
. To compare relevant reference states,
we pursued cryoEM structures of nitrogenase in solution by preparing
an anaerobic work
fl
ow for the generation of single-particle cryoEM
samples within a glove box. Comparisons of datasets with varying
dose, exposure to oxygen, grid types, and conditions were used to
benchmark this procedure (Supplementary Figs. 1, 2). Adsorption of
proteins to the air
water interface (AWI) has long been associated with
apparent discrepancies in expected protein concentration on the grid,
particle orientation bias, and protein denaturation
21
,
22
.Indeed,the
determination of intact MoFe-protein structures required either sur-
factants or the use of carbon-layered grids, due to the localization of
MoFe
As-isolated
to the air AWI, causing strong orientation bias, and dis-
order of its
α
-subunit (Supplementary Fig. 2)
23
25
. Without the use of
surfactants or carbon-layered grids, we observed that the MoFe-
protein adheres to the AWI regardless of ice thickness, and displays
asymmetric loss of density corresponding to residues in the
α
III
domain. The use of cryo-electron-tomography (cryoET) was crucial for
the validation of the MoFe-protein grid and freezing conditions
23
,
24
.
Optimized vitri
fi
cation of the as-isolated MoFe-protein alone
(MoFe
As-isolated
) yielded a 2.13 Å resolution structure in the presence of
detergent (Fig.
1
a and Supplementary Fig. 3).
Structure of the alkaline turnover-inactivated MoFe-protein
Following the determination of the intact MoFe
As-isolated
state, we applied
single particle cryoEM to the high p
H turnover-inac
tivated state,
designated MoFe
Alkaline-inactivated
(Fig.
1
b, c). MoFe-protein was separated
from alkaline acetylene reduction reactions, run either with ATP
(MoFe
Alkaline-inactivated
)orwithoutATP(MoFe
Alkaline
), by size exclusion
chromatography at pH 7.8 (S.E.C.; Supplementary Fig. 4). The
MoFe
Alkaline-inactivated
state displayed a shifted elution time relative to
MoFe
Alkaline
and MoFe
As-isolated
indicating a change in the hydrodynamic
radius of the protein as previously noted
13
. Electron paramagnetic
resonance (EPR) spectroscopy of th
ese states revealed the loss of the
canonical as-isolated
S
= 3/2 signal from the FeMo-cofactor in the
MoFe
Alkaline-inactivated
form (Supplementary Fig. 4). By cryoEM, the control
MoFe
Alkaline
cryoEM structure at 2.14 Å resolution incubated at pH 9.5
closely resembled MoFe
As-isolated
(Fig.
1
bandSupplementaryFig.5).In
contrast, the MoFe
Alkaline-inactivated
cryoEM structure exh
ibited asymmetric
disorder within one
α
-subunit, which likely stems from the averaging of
heterogeneous interme
diate states. This poor density corresponds to
residues 1
48, 354
360, 376
416, and 423
425, which lack traceable
density in the more disjointed
α
-subunit (Fig.
1
c and Supplementary
Fig. 6; 2.37 Å resolution). These
α
III domain residues have been pre-
viouslyshowntorearrangeasseenintheFeMo-cofactor-de
fi
cient
Δ
nifB
MoFe-protein
26
and more recently in cryoEM structures of the nitro-
genase complex
10
,
18
,
19
. It should be noted that in our studies,
similar changes in the
α
-subunit were also observed in the cryoEM
maps of the AWI-perturbed MoFe
As-isolated
protein. However, the
MoFe
Alkaline-inactivated
cryoEM dataset was collected in the presence of
detergent to mitigate this problem, and we directly con
fi
rmed that the
protein was not localized to the AWI as determined by cryoET (Sup-
plementary Fig. 6).
In the MoFe
Alkaline-inactivated
structure, both P-cluster ligands and
one FeMo-cofactor appear well ordered, but within the active site
of the disordered
α
-subunit, the density corresponding to the
crystallographic location of FeMo-cofactor appears distorted (Fig.
2
c).
The coordinating Cys
α
275 loop is near its expected position and
appears to remain coordinated to the distorted cofactor density in that
region (Fig.
2
c). The disordered active site clearly lacks density for
HCA, while the ordered active site shows reduced density at the HCA
coordination site (Fig.
2
c). We developed a method for the quanti
fi
-
cation of HCA extracted from MoFe-protein samples using ion chro-
matography coupled with mass spectrometry (IC-MS), revealing that
while all MoFe
Alkaline
samples analyzed contained approximately the
expected 2 moles HCA per mole MoFe-protein, no HCA was detected
in the MoFe
Alkaline-inactivated
samples (Fig.
2
d). This loss of homocitrate
density in the MoFe
Alkaline-inactivated
maps and the lack of HCA detection by
IC-MS may be due to a chemical alteration of the moiety that results in
a change of its mass and increased positional disorder within the
structure. While the mechanism of this reaction is unknown, we note
that aconitase catalyzes the dehydration and rearrangement of the
hydroxyacid isocitrate at an iron-sulfur cluster
27
. Inductively-coupled
plasma mass spectrometry (ICP-MS) metal analysis of MoFe
Alkaline
and
MoFe
Alkaline-inactivated
demonstrated modest, but statistically insigni
fi
cant,
decreases of 2 moles Fe and 0.4 moles Mo per mole MoFe
Alkaline-inactivated
with respect to the control MoFe
Alkaline
(Fig.
2
e, f).
His-coordination site formation within the high pH turnover-
inactivated state
Within both
αβ
dimers of the MoFe
Alkaline-inactivated
state, Phe
α
300
experiences a rotamer rearrangement with respect to the MoFe
As-isolated
and MoFe
Alkaline
states (Fig.
3
). The movement of this bulky side chain
within the disordered dimer is coupled with altered positioning of three
histidines, His
α
274, His
α
362, and His
α
451, towards a coordination site
for an unidenti
fi
ed ligand ~8 Å from the terminal Fe1 of the FeMo-
cofactor. In addition, while density
around the FeMo-cofactor is poorly
resolved, a rearrangement of the His
α
442 loop that normally ligates the
Article
https://doi.org/10.1038/s41467-023-36636-4
Nature Communications
| (2023) 14:1091
2
FeMo-cofactor can be partially traced into smoothed maps and may
also participate in this coordination in a tetrahedral fashion. Given the
nature of the density and the geom
etry of the coordination, the bound
ligand could be metal. While the conformation of Phe
α
300 is also
altered in the ordered
αβ
dimer of the MoFe
Alkaline-inactivated
protein, His
α
362, His
α
442, and His
α
451residuesinthisorderedsubunitremain
close to their as-isolated positions, precluding the formation of an
equivalent coordination site. Changes in the rotamer state of Phe
α
300,
His
α
274, and His
α
451 were recently described under turnover condi-
tions, pointing to
the possible signi
fi
cance of these residues to substrate
reduction
10
. The additional changes in His
α
362 and His
α
442 found in
MoFe
Alkaline-inactivated
suggest that the formation of a coordination site is a
crucial role for these residue rearrangements and mutations in His
α
274, His
α
362, and His
α
451 correlate to decrea
sed cofactor insertion
and activity
28
,
29
. Thus, the loss of HCA correlates with the disordering of
domain
α
III within the
α
-subunit, distortion of the cofactor density, and
the formation of a new His-coordination site between
α
274, His
α
362,
His
α
451, and His
α
442.
Analysis of the MoFe
Alkaline-inactivated
structure reveals two further
conformational changes in side chains Trp
α
253 and Gln
β
93 with
respect to MoFe
As-isolated
and MoFe
Alkaline
(Supplementary Fig. 7 and
Supplementary Table 2). A distinct Trp
α
253 rotamer was recently
shown to exist under N
2
turnover conditions, and has also been pre-
viously implicated in the control of substrate access to the
active site
10
,
30
. The same conformational change is observed in the
MoFe
Alkaline-inactivated
structure, and may suggest that both acetylene and
N
2
substrates utilize similar substrate access channels. Interestingly,
the Trp
α
253
fl
ip is observed in both
α
-subunits in MoFe
Alkaline-inactivated
,
where previously it was observed only in one subunit under turnover
conditions
10
. Additionally, Gln
β
93 within MoFe
Alkaline-inactivated
experi-
enced a
fl
ip away from its resting state position in the
β
-subunit adja-
cent to the disordered
α
-subunit. This residue has been suggested to
be along the path of egress for the NH
3
product, and is also positioned
close to the P-cluster
31
.
Three-dimensional variability analysis (3DVA) of the
MoFe
Alkaline-inactivated
cryoEM structure revealed the ordering and dis-
ordering of the
α
-subunits (Supplementary Movie 1), indicating the
fi
nal reconstruction contains a mixture of fully ordered, partially dis-
ordered, and fully disordered
α
-subunits
32
. These changes appear
correlated with a breathing motion within the
α
-subunit. A morph
Fig. 1 | Anaerobic cryoEM structures of MoFe-protein. a
The 2.13 Å resolution
cryoEM map of MoFe
As-isolated
puri
fi
ed from
A. vinelandii
.
b
The 2.14 Å resolution
cryoEM map of MoFe
Alkaline
from a control acetylene reduction reaction performed
at pH 9.5 without ATP isolated via S.E.C. conducted at pH 7.8, demonstrating the
same overall architecture as MoFe
As-isolated
.
c
The 2.37 Å resolution cryoEM map of
MoFe
Alkaline-inactivated
from an acetylene reduction reaction performed at pH 9.5 with
ATP isolated via S.E.C. conducted at pH 7.8, displaying asymmetric disorder within
the
α
-subunit density.
d
f
Location of disorder within the
α
III domain of the
α
-
subunit in the
d
MoFe
As-isolated
,
e
MoFe
Alkaline
,and
f
MoFe
Alkaline-inactivated
structures.
α
-
subunits are illustrated in blue and
β
-subunits are illustrated in yellow. Structures
were solved in the presence of CHAPSO to prevent interactions with the air
water
interface.
Article
https://doi.org/10.1038/s41467-023-36636-4
Nature Communications
| (2023) 14:1091
3
between the MoFe
Alkaline-inactivated
and MoFe
As-isolated
structures illustrates a
similar movement, with shifts apparent in
α
III domain helices Pro
α
302
Lys
α
315, Glu
α
318
Arg
α
345, and Lys
α
426
Met
α
434, and
β
-
strand Lys
α
349
Ile
α
355 (Supplementary Movie 2). Intermediate
subsets corresponding to the variably ordered states identi
fi
ed in
3DVA were distinguishable across
fi
ve maps with either two disordered
α
-subunits or asymmetrically disordered
α
-subunits (Supplementary
Fig. 8). Models built into these intermediate MoFe
Alkaline-inactivated
maps
highlight several features. All maps lack HCA density, emphasizing the
consistent loss of this ligand. Additionally, as the order increases
within one of the
α
-subunits, cofactor density order is regained, Phe
α
300 returns to its resting state position, and the His-coordination site
is lost.
The homocitrate-de
fi
cient MoFe
Δ
NifV
structure resembles the
MoFe
Alkaline-inactivated
state
To determine whether the features of the MoFe
Alkaline-inactivated
state
stem solely from the loss of HCA, we characterized the
homocitrate-de
fi
cient MoFe-protein puri
fi
ed from the homo-
citrate synthase (
nifV
) deletion strain of
A. vinelandii
(MoFe
Δ
NifV
)
33
.
Like MoFe
Alkaline-inactivated
,MoFe
Δ
NifV
showed similar SEC shifts in
elution time relative to MoFe
As-isolated
and had diminished levels of
acetylene reduction (Supplementary Fig. 9). Likewise, EPR spec-
troscopy revealed a loss of the canonical as-isolated
S
=3/2signal
from the FeMo-cofactor in the MoFe
Δ
NifV
form, comparable to the
MoFe
Alkaline-inactivated
state. The single particle cryoEM structure of
MoFe
Δ
NifV
also showed asymmetric disordering of residues 14
19,
25
26, and 408
417 in one of the
α
-subunits (Fig.
4
aandSup-
plementary Fig. 10). No density was observed at the canonical
HCA position within the active site of the disordered
α
-subunit,
but a smaller density remains in the HCA pocket within the cor-
responding ordered
α
-subunit (Fig.
4
b). No homocitrate was
detected by IC-MS, but citrate was isolated from the MoFe
Δ
NifV
sample at a molar ratio of 0.2-mole citrate per mole MoFe
Δ
NifV
.For
comparison, a reduced occupancy of citrate (50%) was previously
reported in
Δ
NifV MoFe-protein isolated from
Klebsiella pneu-
moniae
(PDB code 1H1L
34
;Fig.
4
c). Variations in citrate incor-
poration between this study and ours may stem from differences
between the bacteria or growth media, but in both cases, sub-
stoichiometric levels of citrate were observed.
The
K. pneumoniae
Δ
nifV crystal structure also lacked the disorder
within one
α
-subunit, perhaps suggesting that its crystal lattice favored
more ordered states of the
α
-subunit. In our work, ~39 moles Fe and 1.8
Mo per molecule of MoFe
Δ
NifV
were detected by ICP-MS of the puri
fi
ed
protein, suggestive of a full complement of clusters (Fig.
4
d). The Phe
α
300 side chain is
fl
ipped in the disordered
α
-subunit of MoFe
Δ
NifV
,
however, His
α
362, His
α
442, and His
α
451 remain close to their resting
state positions in both dimers, preventing the formation of the histi-
dine coordination site. Subsets of these MoFe
Δ
NifV
particles isolated by
3DVA did not demonstrate an equivalent coordination site (Supple-
mentary Fig. 8). In addition, while Gln
β
93 remained close to the as-
isolated position in MoFe
Δ
NifV
,Trp
α
253 was observed to undergo the
same conformational change as observed in the MoFe
Alkaline-inactivated
state in its more ordered
α
-subunit (Supplementary Fig. 7 and Sup-
plementary Table 2).
Structure of a previously uncharacterized MoFe
Δ
NifV
-NafT com-
plex revealed by cryoEM
Further data collection and classi
fi
cation of a subset of MoFe
Δ
NifV
par-
ticles resulted in a 2.71 Å overall resolution map with a protruding
density that we hypothesized belonged to an endogenous binding
partner pulled down with MoFe
Δ
NifV
during puri
fi
cation (Fig.
5
aand
Supplementary Fig. 11). This density appears with more disordered
MoFe
Δ
NifV
particles (Supplementary Table 2 and Supplementary Fig. 8).
A partial model built into this additional density was cross-referenced
against Alpha Fold-predicted models of proteins identi
fi
ed in puri
fi
ed
MoFe
Δ
NifV
samples by proteomic analysis
35
37
. This approach revealed
that the AlphaFold model of an uncharacterized protein of 15 kDa, a
product of the Avin_01560 gene on the
nif
operon, matched the
extended density (Fig.
5
a). This gene product has also been annotated
as the nitrogenase-associated factor protein T (NafT) in previous
studies
14
,
15
. Building the model into this density yields the MoFe
Δ
NifV
-
NafT complex (Fig.
5
a, b). To distinguish NafT from the MoFe-protein
α
-and
β
-chains, we refer to this subunit as the epsilon (
ε
)chainwhen
designating residues. There is greater relative disorder and lower
resolution within the region of the NafT, likely due to residual het-
erogeneity in the particles. The
binding site for this protein on
MoFe
Δ
NifV
partially overlaps with that of the nitrogenase Fe-protein in
the MoFe-protein:Fe-protein ADP-AlF
4
-
stabilized complex and buries
Fig. 2 | Structure and composition of the alkaline turnover-inactivated MoFe-
protein active site. a
CryoEM density carved around the FeMo-cofactor at 7
σ
in
MoFe
As-isolated
puri
fi
ed from
A. vinelandii
demonstrating the presence of the FeMo-
cofactor coordinated by Cys
α
275, His
α
442, and an HCA in both
αβ
dimers.
b
CryoEM density carved around the FeMo-cofactor at 7
σ
in MoFe
Alkaline
isolated
from a control acetylene reduction reaction mixture at pH 9.5 without ATP,
demonstrating the maintenance of the FeMo-cofactor coordinated by Cys
α
275,
His
α
442, and an HCA in both
αβ
dimers.
c
CryoEM density carved around the
FeMo-cofactor at 7
σ
in MoFe
Alkaline-inactivated
isolated from an acetylene reduction
assay at pH 9.5 with ATP, demonstrating the presence of the FeMo-cofactor
coordinated by Cys
α
275, His
α
442, and an unknown ligand (UNX) in the HCA site
in the ordered
αβ
dimer (top panel). The loss of recognizable density for the
FeMo-cofactor and the HCA is evident in the disordered
αβ
dimer (lower panel).
d
Quanti
fi
cation of homocitrate by an IC-MS method developed for this work
illustrates the approximately expected 2 moles HCA per mole of tetrameric
protein in MoFe
Alkaline
(blue circles), however, HCA was not detected (n.d.) within
the MoFe
Alkaline-inactivated
samples (red circles), which have been represented as zero
values in this data.
e
ICP-MS metal quanti
fi
cation of Fe illustrates a decrease from
the mean of 30.7 moles Fe per mole protein in MoFe
Alkaline
to a mean of 28.7 moles
Fe per mole tetrameric protein in MoFe
Alkaline-inactivated
.
f
ICP-MS metal quanti
fi
cation
of Mo shows a decrease from the mean of 1.79 moles Mo per mole protein
in MoFe
Alkaline
to a mean of 1.35 moles Mo per mole tetrameric protein in
MoFe
Alkaline-inactivated
. Each data point in panels
d
f
represents a protein isolated
from an independent reaction and columns are shown as the mean ± sd. Statis-
tical analyses of three distinct replicates shown in panels
d
f
used paired
t
-tests
and two-tailed
P
values greater than 0.05 were considered not signi
fi
cant (n.s.).
Source data are provided as a Source Data
fi
le. HCA
R
-Homocitrate, Fe iron,
Mo molybdenum.
Article
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Nature Communications
| (2023) 14:1091
4
approximately ~1,400 Å
2
surface area (PDB 1N2C
38
;Fig.
5
c, d). At the
interface between the two proteins, a well-ordered amphipathic helix
from Trp
ε
117 to Ile
ε
131 packs against the MoFe
Δ
NifV
protein
α
-and
β
-
subunits. Additionally, a loop in NafT spanning residues Lys
ε
10 to Leu
ε
17 extends into the MoFe
Δ
NifV
α
-subunit. This extension allows for
hydrogen bonding between Ser
ε
14 and Tyr
α
281, and a salt bridge
between Arg
ε
16 and Asp
α
200. Perhaps more strikingly, this loop adds
two histidines (His
ε
9andHis
ε
11) to a cluster of histidines that includes
His
ε
95, His
α
195, and His
α
196, four of which are situated within 10 Å
of each other (Fig.
5
d). It is tempting to speculate that under certain
conditions this histidine cluster may be capable of metal or cluster
binding. While the function of the NafT protein has yet to be eluci-
dated, this structure demonstrates that it recognizes homocitrate-
de
fi
cient forms reminiscent of the MoFe
Alkaline-inactivated
state.
In the MoFe
Δ
NifV
-NafT state, the MoFe
Δ
NifV
shows asymmetric dis-
ordering of residues 1
48, 376
383, 390
398, and 402
409 in one of
its
α
-subunits (Fig.
5
). NafT was also disordered from residue 41
48.
The Phe
α
300 side chain is
fl
ipped in both
α
-subunits. In the ordered
subunit, His
α
274, His
α
362, and His
α
451 remain close to their resting
state positions. By contrast, in the disordered subunit in concert with
the movement of Phe
α
300, His
α
362, and His
α
451 have modi
fi
ed
positions, appearing to form a His-triad site with His
α
274. This site
MoFe
M
o
F
e
As-isolated
A
s
-
i
s
o
l
a
t
e
d
a
b
c
Ordered
Disordered
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
MoFe
M
o
F
e
Alkaline
A
l
k
a
l
i
n
e
MoFe
M
o
F
e
Alkaline-inactivated
A
l
k
a
l
i
n
e
-
i
n
a
c
t
i
v
a
t
e
d
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
7.7 Å
7
.
7
Å
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
451
4
5
1
His
H
i
s
α
442
4
4
2
His
H
i
s
α
274
2
7
4
His
H
i
s
α
362
3
6
2
FeMo-cofactor
F
e
M
o
-
c
o
f
a
c
t
o
r
Cys
C
y
s
α
275
2
7
5
MoFe
M
o
F
e
As-isolated
A
s
-
i
s
o
l
a
t
e
d
d
HCA
H
C
A
MoFe
M
o
F
e
Alkaline-inactivated
A
l
k
a
l
i
n
e
-
i
n
a
c
t
i
v
a
t
e
d
Fig. 3 | His-coordination site formation within the high pH turnover-
inactivated state. a
His
α
274, His
α
362, and His
α
451 within MoFe
As-isolated
puri
fi
ed
from
A. vinelandii
. The upper panel displays the His-coordination site in the more
ordered dimer, while the lower panel displays the more disordered dimer.
b
His
α
274, His
α
362, and His
α
451 in MoFe
Alkaline
from an acetylene reduction reaction
performed at pH 9.5 without ATP isolated via S.E.C. conducted at pH 7.8. The upper
panel displays the His-coordination site in the more ordered dimer, while the lower
panel displays the more disordered dimer.
c
His
α
274, His
α
362, and His
α
451 in
MoFe
Alkaline-inactivated
from an acetylene reduction reaction performed at pH 9.5 with
ATP isolated via S.E.C. conducted at pH 7.8. The upper panel displays the His-
coordination site in the more ordered dimer, while the lower panel displays the
more disordered dimer. The repositioning of the His
α
442 side chain is evident in
the disordered dimer.
d
Enlarged overlay of the active sites and His-coordination
site from the disordered dimers of MoFe
As-isolated
(yellow) and MoFe
Alkaline-inactivated
(blue). All densities shown are carved around the atoms at 7
σ
.HCA
R
-Homocitrate.
Article
https://doi.org/10.1038/s41467-023-36636-4
Nature Communications
| (2023) 14:1091
5
differs from that in the MoFe
Alkaline-inactivated
state in the absence of His
α
442 (Supplementary Table 2). In both
α
-subunits, Trp
α
253 has the
same altered conformation as that observed in MoFe
Alkaline-inactivated
,but
Gln
β
93 remains in the as-isolated state (Supplementary Fig. 7).
Discussion
The features observed in this study, including HCA loss, distorted
cofactor density,
α
-subunit disordering, the formation of a His-
coordination site within the MoFe
Alkaline-inactivated
protein, and the
recognition of the similar MoFe
Δ
NifV
state by the endogenous binding
partner NafT, have important implications for the mechanism of
nitrogenase. Based upon these observations, summarized in Supple-
mentary Table 2, we suggest that the bidentate coordination of the
HCA acts in part as a staple that helps maintain the FeMo-cofactor
within the binding pocket. Perturbations in the cofactor site associated
with HCA loss are correlated with disordering in the
α
-subunit as seen
in the MoFe
Alkaline-inactivated
and MoFe
Δ
NifV
structures. The asymmetry
observed in both the MoFe
Alkaline-inactivated
and MoFe
Δ
NifV
structures may
suggest crosstalk between the two dimers
10
,
39
,
40
, though the 3DVA
results of the MoFe
Alkaline-inactivated
structure also indicate that both
dimers can be either ordered or disordered, as well. Our cryoEM
structures further support the possibility of altered coordination or
even complete dissociation of HCA and His
α
442 from Mo during
turnover, enabling repositioning of the FeMo-cofactor while it remains
tethered to the protein through Cys
α
275. This accounts for the dis-
ordered density observed in the active sites of our MoFe
Alkaline-inactivated
and MoFe
Δ
NifV
cryoEM maps. A potential driving force for this trans-
formation could be the preference for hydroxytricarboxylic acid spe-
cies such as HCA to form tridentate metal complexes
41
,withthe
binding of a second HCA carboxyl group to Mo coupled to the
displacement of the His
α
442 ligand. One consequence of a tri-
dentate coordination mode would be to enable rotation of the cofac-
tor about the Fe1
Cys
α
275 bond, thereby providing a rationalization
for the apparent interconversion of belt sulfur positions under
turnover
42
,
43
. Rotation of the cofactor would avoid the high energetic
barrier to internal scrambling noted by Dance
44
. Further, the
His-coordination site observed within the turnover-inactivated
MoFe
Alkaline-inactivated
structure may serve to usher the mobile cluster
towards the surface of the protein, either for the expulsion of a
damaged state or for catalysis at the surface of the MoFe-
protein (Fig.
5
d).
While the inactivation reaction and subsequent features of
MoFe
Alkaline-inactivated
described take place at high pH, these processes are
also expected to occur at physiological pH albeit at a decreased rate
13
.
During slow diazotrophic growth, inactivated MoFe-protein should
accumulate, thus highlighting the possible need for a repair mechan-
ism. In vivo disordering, rearrangements observed in the active site-
adjacent histidines, and cofactor mobility may recruit binding partners
such as NafT and could constitute a mechanism for recognizing
damaged states. In other cellular redox systems, such as photosystem
II, auxiliary proteins can facilitate the formation of multisubunit
complexes or the insertion of metallocofactors. Thus, it is possible that
NafT similarly serves as an assembly factor for the recruitment of
additional proteins for stabilization, repair, degradation, or catalysis.
Given the similarities between the MoFe
Δ
NifV
and MoFe
Δ
NifB
states, it is
also possible that this protein plays a role in the biogenesis of nitro-
genase or cofactor insertion. Further studies are being conducted to
understand the relationship between the features observed in the
captured MoFe
Alkaline-inactivated
state, the fate of homocitrate, and the role
of NafT in nitrogenase function.
Methods
Puri
fi
cation and characterization of MoFe
As-isolated
and MoFe
Δ
NifV
The MoFe
As-isolated
and the Fe-protein
As-isolated
were puri
fi
ed from
wild-type
A. vinelandii
Lipman (ATCC 13705, strain designation
OP). MoFe
Δ
NifV
was puri
fi
ed from a
nifV
deletion strain, DJ605,
which was a kind gift from Dr. Dennis Dean
33
.Puri
fi
cations were
performed under anaerobic conditions using a combination of
Schlenk line techniques and anaerobic chambers with oxygen-
scrubbed argon
13
. Protein concentrations were determined by
amino acid analysis at the UC Davis Molecular Structure Facility.
Concentrations in mg/mL were calculated based on molecular
weights of 230 and 64 kDa for the MoFe-protein and Fe-protein,
respectively. The purity of samples were assessed by bottom-up
0.0
0.1
0.2
0.3
0
10
20
30
40
50
MoFe
M
o
F
e
ΔNifV
Δ
N
i
f
V
Ordered
O
r
d
e
r
e
d
Disordered
D
i
s
o
r
d
e
r
e
d
a
b
90°
His
H
i
s
α
442
4
4
2
Cys
C
y
s
α
442
4
4
2
Citrate
C
i
t
r
a
t
e
His
H
i
s
α
442
4
4
2
Cys
C
y
s
α
442
4
4
2
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
His
H
i
s
α
451
4
5
1
His
H
i
s
α
362
3
6
2
His
H
i
s
α
274
2
7
4
Phe
P
h
e
α
300
3
0
0
His
H
i
s
α
442
4
4
2
c
d
e
[HCA]
[CA]
[Fe]
[Mo]
Moles acid/mole MoFe
ΔNifV
Moles metal/mole MoFe
ΔnifV
FeMo-cofactor
F
e
M
o
-
c
o
f
a
c
t
o
r
His-coordination site
H
i
s
-
c
o
o
r
d
i
n
a
t
i
o
n
s
i
t
e
Ordered
O
r
d
e
r
e
d
Disordered
D
i
s
o
r
d
e
r
e
d
n.d.
Fig. 4 | The homocitrate-de
fi
cient MoFe
Δ
NifV
resembles the MoFe
Alkaline-inactivated
state, with partial occupancy citrate replacing homocitrate in the active site.
a
2.58 Å resolution cryoEM map of MoFe
Δ
NifV
puri
fi
ed from
A. vinelandii
lacking a
functional
nifV
gene product.
b
CryoEM density carved around the FeMo-cofactor
at 7
σ
in MoFe
Δ
NifV
demonstrating the presence of the FeMo-cofactor coordinated
by Cys
α
275, His
α
442, and citrate in place of the HCA site in the ordered
αβ
dimer
(top panel). A considerable loss of recognizable density for the FeMo-cofactor and
the HCA is evident in the disordered
αβ
dimer (bottom panel).
c
Quanti
fi
cation by
an IC-MS method developed for this work con
fi
rms the presence of citrate (CA; blue
circles) within the puri
fi
ed MoFe
Δ
NifV
protein at very low levels (0.2 moles citrate per
mole protein). HCA was not detected (n.d.) in this sample (red circles).
d
ICP-MS
metal quanti
fi
cation shows ~39 moles iron (Fe; orange circles) and ~1.8 moles
molybdenum (Mo; teal circles) per mole puri
fi
ed MoFe
Δ
NifV
protein, respectively.
e
CryoEM density carved at 7
σ
around His
α
274, His
α
362, and His
α
451 in MoFe
Δ
NifV
equivalent to the coordination site observed in MoFe
Alkaline-inactivated
.Inpanels
c
,
d
,
n
= 3 independent reads of the same sample and columns are shown as the
mean ± s.d. Source data are provided as a Source Data
fi
le. HCA
R
-Homocitrate, CA
citrate, Fe iron, Mo molybdenum.
Article
https://doi.org/10.1038/s41467-023-36636-4
Nature Communications
| (2023) 14:1091
6