Mario Alberto Hernndez Salinas.

13090338

Nitrogenase FeMo cofactor: an atomic structure

in three simple steps.
The enzyme nitrogenase catalyzes the six-electron reduction of molecular
dinitrogen to ammonium, concomitant with the reduction of protons to yield
hydrogen gas.
This review focuses on the history of the structure determination of FeMo
cofactor, a process that extended over two decades and involved several
iterations and corrections that are unusual and unexpected within the
stablished field of X-ray crystallography.
Bioinorganic metal centers are large, often asymmetric, and they may require a
small army of maturation factors for their assembly. Yet, only the synergies of
the inorganic metals site together with a highly evolved protein ligand will give
rise to the emergent properties that endow such proteins with catalytic
capacities and efficiencies that we consistently fail to reproduce in the lab.
A crucial step in understanding the function of a metal-containing enzyme is
thus always obtain a glimpse of its three-dimensional structure.
Nitrogenase is the only known enzyme able to break the triple bond of the
dinitrogen molecule, whichwith a bond energy of -946 kJ mol-1is the
stablest bond any biological system has to break.
An unmatched wealth of physiological, biochemical, spectroscopic, and
theoretical data has been accumulated, to the point that almost every possible
reaction pathway has already been postulated. This may well include the
correct one, but there remains quite some lack of agreement in the field as to
which one that is.
The story of the structure of FeMo cofactor is an unusual example of the power
of chemical analysis, and is also an example of the unexpected pitfalls that
may come with it.

Step 1: the structure of FeMo cofactor

FeMo cofactor contains one molybdenum and seven iron ions. The cluster was
described as being composed of two distinct subclusters, one has 1 Mo, 3 Fe,
and 3S; and the other one has 4 Fe and 3S.
They were bridged by ligands assigned as two sulfides and a third, unidentified
ligand X that soon thereafter was found to be another sulfide, this large
metal cluster contained an apparently empty central cavity.
. It was surrounded by six iron atoms, Fe2Fe7, arranged as a trigonal prism,
each of which was coordinated exclusively to three of the sulfides, leaving a
fourth coordination site open to complete a tetrahedral geometry, and this

fourth site was directed into the central cavity. The triangular sides of the iron
prism were capped by the seventh iron atom, Fe1, on one side, and the
molybdenum ion on the other, creating a complex (Mo, 7 Fe, 9 S] structure with
intrinsic threefold symmetry.
The six central iron cations would tend to strongly repel each other in any
energy minimization approach, and in order to reproduce the experimentally
observed structure, this generally had to be overcome by postulating strong
metalmetal interactions within the trigonal prism. The assumption had the
further advantage that it provided a rationale for the absence of any ligand
binding, as it essentially described the core of FeMo cofactor to be a block of
solid metal.

Step 2: something missing in the middle.

In the following years, structural analyses of MoFe protein were presented for
two further organisms, Clostridium pasteurianum and Klebsiella pneumoniae,
and in both the exact same structure of FeMo cofactor was observed.
At 1.16 A level of detail, electron density maps show distinct maxima for
individual atoms that allow a precise positioning of noncanonical moieties such
as metal clusters. In the case of MoFe protein, however, the effect of higher
resolution was unprecedented, in that it revealed the entirely unexpected
presence of a light atom in the center of the cluster, within the trigonal prism
formed by irons Fe2Fe7. Further calculations showed that the electron density
maximum for this atom disappeared at resolutions lower than 1.55 A.
Fourier transforms such as in the generation of a spatial electron density
distribution map in crystallography involve the integration of data points in
frequency space that is represented by periodic functions. A complete
Fourier transform will yield an exact reproduction of an object in real space,
but any lack of data will not lead to gaps or incompleteness after
transformation, but rather will lead to an overall deterioration of the quality of
the electron density map.
An obvious candidate atom for an ironsulfur cluster was one from a further
sulfide, but this possibility was quickly ruled out for two reasons. Firstly, the
bond distance of 2.0 A was too short for an FeS bond, which commonly
averages around 2.22.3 A, and secondly, the observed electron density was
clearly insufficient to represent a sulfur species, limiting the search for likely
candidates to the light elements carbon, nitrogen, and oxygen.
The synthesis of a cobalt carbonyl complex with exactly the same geometry
that also contained interstitial nitrogen was reported in 1979, followed by
similar models with carbon and oxygen as central atoms, respectively.
From a functional perspective, however, the question of the nature of the
central atom was fundamental, as it seemed difficult to envision a mechanism
that would lead to the inclusion of a nitrido species and that was not in some

way connected to the catalytic activity of nitrogenase, the cleavage of the N2

molecule.
It was found that the center if FeMo cofactor is C. [1]

Step 3: the nature of X

The missing piece at this point was positive evidence concerning the nature of
the central ligand. Three independent analyses had thus identified carbon as
the most likely candidate, each providing data based on direct observation of
the signature of the interstitial light atom. This set of data not only settled the
question concerning the nature of this atom, but also provided a crucial clue
with regard to its origin: single-carbon unit transfer in biochemical systems is
well known and characterized, and living organisms generally use one of three
possible systems for this type of chemistry.
The quest for the identity of the central ligand in FeMo cofactor had thus come
to an end, and we now know that nitrogenase carries a core of carbon-doped
iron with a hint of molybdenuma heart of steel.
References
https://www.youtube.com/watch?v=6nM-8ZhUIQ8
(Einsle, 2014)