Cleaving the N,N Triple Bond: The Transformation of Dinitrogen to Ammonia by Nitrogenases - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 148

X-ray crystal structures of both the nucleotide-free and the MgADP-bound forms of
the Fe protein have been solved (Figure 2a )[ 29 ], the structure of the MgATP-bound
form of this protein is still unavailable. Nevertheless, biochemical and mutagenetic
studies have revealed the potential involvement of a so-called P-loop at the Walker
A motif in the binding of MgATP to the Fe protein [ 6 , 10 , 27 , 30 - 32 ]. The P-loop
features Ser 16 , which coordinates a Mg 2+ ion and a conserved Lys 10 residue through
its hydroxyl group, thereby forming a salt bridge with the

ʲ

-phosphate of the
nucleotide and consequently locking the nucleotide in its binding site (Figure 2a, b ).
Upon ATP binding, the Fe protein undergoes a conformational rearrangement
that renders its [Fe 4 S 4 ] cluster more exposed to solvent and, consequently, there is
an increase in the reactivity of the [Fe 4 S 4 ] cluster toward metal chelators [ 33 ].
Consistent with this observation, electron paramagnetic resonance (EPR) analysis
also suggests a potential change of the environment of the [Fe 4 S 4 ] cluster in the Fe
protein upon incubation with MgATP [ 33 - 36 ]. Interestingly, the crystal structures
of the Fe protein show that the nucleotide-binding sites are 15-19

away from the
[Fe 4 S 4 ] cluster [ 4 , 6 , 9 , 29 ]. The lack of direct contact between the nucleotide- and
cluster-binding sites provides further support for the proposed conformational
change of the Fe protein upon MgATP binding that alters the location of the
[Fe 4 S 4 ] cluster [ 4 ]. While the structural information of such a conformational
rearrangement has not been acquired so far, there is indirect evidence from muta-
genic studies that certain site-directed variants of the Fe protein cannot undergo
conformational changes due to the perturbation of the signal transduction between
the nucleotide- and cluster-binding sites [ 31 , 32 ]. Further investigations of these
variants suggest that the Walker A motif, the so-called Switch I (residues 38-44)
and Switch II (residues 125-129) regions, and a conserved region around the
residue Ala 157 are all crucial for the MgATP-dependent protein conformational
changes of the Fe protein (Figure 2b )[ 4 , 6 , 10 , 27 ]. Based on these results, as well
as the crystal structure of the MgADP-bound Fe protein, it has been proposed that
(i) Switch I acts as a direct link that communicates between the nucleotide-binding
site and the Fe protein/MoFe protein docking interface; and (ii) Switch II mediates
signal transduction between the nucleotide-binding site and the [Fe 4 S 4 ] cluster
[ 29 , 37 ]. The purpose of this MgATP binding-induced change in protein confor-
mation is still under debate; however, this conformational change is clearly essen-
tial for catalysis, as any mutation that prevents it from occurring renders the Fe
protein inactive in substrate reduction.

Å

2.1.2 The [Fe 4 S 4 ] Cluster

The [Fe 4 S 4 ] cluster is symmetrically ligated by two cysteine residues, Cys 97 and
Cys 132 , from each subunit of the Fe protein (Figure 2c ). It has a +1 oxidization state
when the protein is isolated in excess dithionite. In this state, the cluster displays a
mixed electronic spin state of S ¼ 1/2 and S ¼ 3/2 [ 38 , 39 ]. The distribution of