INSIGHTS | PERSPECTIVES
1568 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
Even though nitrogen makes up almost 80% of the atmosphere, it is a limiting nutrient for biomass production. The low reactivity of nitrogen gas (N2) is a result of its very strong, unpolarized triple bond. Nitrogenase is the only
enzyme known that can break this bond to
produce compounds such as ammonia (NH3)
for use in biosynthetic pathways. The atomic
structure of this amazing system has been
known for more than two decades (1, 2), but
the chemical mechanism of this central reaction remains unknown. In a biochemical
and structural tour de force, on page 1620 of
this issue, Spatzal et al. (3) report the crystal
structure of carbon monoxide (CO) bound
to the catalytic metal cluster of the enzyme.
This work revealed an unexpected structural
rearrangement of the cofactor.
The industrial equivalent of the nitrogenase reaction is the Haber-Bosch process
developed during the first decades of the
20th century (4). In this process, N2 is reacted with H2 to produce NH3. The overall
reaction is energetically favorable but has a
very high activation barrier. In the Haber-Bosch process, this barrier is overcome with
an iron-based inorganic catalyst at high
temperatures and pressures (400°C and
Whereas the Haber-Bosch process uses
a big hammer to break the triple bond, nitrogenase uses a much more elegant and
complicated catalytic tool to disassemble
N2 at ambient temperature and pressure.
Enzymes commonly use transition metal
ions to perform the most challenging redox
reactions. Nitrogenase houses a dauntingly
complicated cofactor consisting of no less
than one molybdenum, seven iron, and nine
sulfur ions, all beautifully organized around
a central carbon. It was only a few years ago
that the identity of the central atom was finally determined (5, 6).
Although we know what this complicated
tool does and looks like, we have not been
able to understand how it functions or to
obtain snapshots of it in action. A number
of mechanistic proposals have been made
that suggest either molybdenum or iron as
the central catalytic element. Thus, even the
key clue of which part of the cofactor di-
rectly interacts with the substrate has been
missing (7). This issue is commonly studied
by determining structures of the protein co-
factor with various ligands bound, such as
inhibitors or substrate analogs. Carbon mon-
oxide (CO) is a particularly attractive ligand
to obtain insight into nitrogenase chemis-
try. It is a diatomic neutral molecule that is
isoelectronic with N2. It also functions as a
reversible inhibitor of N2 reduction and can
even act as a substrate and be chemically
modified by the enzyme, albeit very slowly.
Such experiments have proven very difficult to perform. To bind any ligand, the
cofactor needs to be in its reduced form.
Nitrogenase will only accept its natural re-ductant, an oxygen-sensitive iron-sulfur protein. Also, CO will only bind to the partially
reduced protein generated in a reaction mixture during enzymatic turnover, conditions
opposite to the very pure protein solutions
usually required for crystallization. This resulted in a wall of technical obstacles that
had prevented determination of complex
structures and a detailed structural understanding of the mechanism.
Spatzal et al. overcame these obstacles
with a number of elegant procedures—for
example, by succeeding to rapidly crystallize
the protein directly from enzymatic reaction
mixtures. The structure provided a great surprise in that CO binding actually reorganizes
the cofactor structure. X-ray anomalous-scattering measurements show that one of
the sulfur atoms is removed from the cluster.
This vacancy opens a new binding position
on the cofactor where a sub-
strate can coordinate two iron
ions simultaneously in a bridg-
ing manner. Carbon monoxide
binds in this position, where it
interacts with both iron ions,
and it is also in close proximity
to the central carbon atom of the
The direct interaction with
iron indicates that there may
be mechanistic similarities to
the iron-catalyzed Haber-Bosch
reaction. Interestingly, the di-iron bridging position also has
similarities to substrate-binding
modes proposed in other enzymes performing—for example,
Together, the results suggest that the
previously available picture of the cofactor
actually shows a tool protected for storage
rather than ready to perform its function.
The mode of N2 binding cannot be conclusively assigned, but the studies of Spatzal et
al. establish the structurally dynamic nature
of the cofactor. The study does not give a
complete and final structural description of
nitrogenase catalysis, but it does provide the
first crack in the wall. ■
REFERENCES AND NOTES
1. J. Kim, D. C. Rees, Science 257, 1677 (1992).
2. J. Kim, D. C. Rees, Nature 360, 553 (1992).
3. T. Spatzal et al ., Science 345, 1620 (2014).
4. V. Smil, Enriching the Earth: Fritz Haber, Carl Bosch, and
the Transformation of World Food Production (MIT Press,
Cambridge, MA, 2004).
5. K. M. Lancaster et al., Science 334, 974 (2011).
6. T. Spatzal et al ., Science 334, 940 (2011).
7. B. M. Hoffman, D. Lukoyanov, Z.-Y. Yang, D. R. Dean, L. C.
Seefeldt, Chem. Rev. 114, 4041 (2014).
8. M. R. A. Blomberg, T. Boro wski, F. Himo, R.-Z. Liao, P. E. M.
Siegbahn, Chem. Rev. 114, 3601 (2014).
9. M. H. Sazinsky, S. J. Lippard, Acc. Chem. Res. 39, 558
10. C. E. Tinberg, S. J. Lippard, Acc. Chem. Res. 44, 280 (2011).
11. D. Lundin, A. M. A. Poole, B.-M. B. Sjöberg, M. Högbom, J.
Biol. Chem. 287, 20565 (2012).
12. R. H. Holm, P. Kennepohl, E. I. Solomon, Chem. Rev. 96,
Supported by the Swedish research council and the Knut and
Alice Wallenberg Foundation.
A dynamic tool for nitrogen reduction
By Martin Högbom
Carbon monoxide reveals new possibilities for substrate binding in nitrogenase
Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden. E-mail: firstname.lastname@example.org
Uncovered potential. The catalytic Mo/Fe/S metal cluster of
nitrogenase is shown (molybdenum in turquoise, iron in orange, carbon
in gray, and sulfur in yellow). Binding of carbon monoxide rearranges the
cluster, revealing new possibilities for substrate binding and reduction.