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 a-His195 (2.8 Å, NE2–OC distance) and
a-Val70 (3.4 Å, closest methyl–OC distance).
Modifications to both side chains were reported
to substantially alter the catalytic properties
of the enzyme. An a-His195 to a-Gln195 mutation resulted in the loss of N2 reduction activity,
while an a-Val70 to a-Ala/Gly70 alteration was
reported to enable the reduction of longer carbon-chain substrates and highlighted the potential involvement of Fe6 in substrate reduction
(29–33). In the structure presented here, a-His195
is in hydrogen bonding distance to the oxygen
of CO, and a-Val70 directly flanks the binding
site (Fig. 1D).
The displacement of S2B could be facilitated
by a proton donation from a-His195 to yield
either HS– or H2S, which would generate a better
leaving group than S2–. Although the dissociation of a sulfur may seem surprising, it opens
up the ligand binding site, because the large
radius of S2– effectively shields the cofactor Fe
atoms in the resting state from substrate and/or
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 toward 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 scaf-
fold 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 (Fig. 1C). The modest adjustments of the
remaining scaffold upon CO binding are sug-
gestive of an important role for the interstitial
carbon in stabilizing the cofactor during re-
arrangements and substitutions to the coordi-
nation 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. Note that 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
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 N2, 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 emphasize the
relevance of the CO-bound structure toward understanding dinitrogen binding and reduction.
This sheds light on N2 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.
1622 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
Fig. 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 (2Fobs – Fcalc) map
is contoured at 4.0 s 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 s, showing the presence of anomalous density at the S2B site. Color scheme is according
to Fig. 1.
Fig. 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 a
and b subunits of the a2b2 MoFe-protein. The potential SBS is located 22 Å away from its former position in
the FeMo-cofactor (S2B-site). (B) Close-up view of 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 s, showing the presence of anomalous density at the
potential SBS. The side-chain sulfur of a-Met112 provides an internal standard for full occupancy. Alternate
conformations for b-Arg453 are indicated. The color scheme is according to Fig. 1.