passes through the P-cluster and finally reaches the M-cluster where substrates are
reduced. Remarkably, this process repeats itself multiple times to successively
accumulate sufficient amounts of reducing equivalents for the reduction of N 2 or
other substrates [24–26].
2.2 Structures and Properties of P- and M-Cluster
of Mo-Nitrogenase
Regarded among the most complex metalloclusters in nature, both the P- and the
M-cluster of Mo-nitrogenase can actually be viewed as fused [Fe 4 S 4 ]-like modules.
The [Fe 8 S 7 ] architecture of the P-cluster is essentially two [Fe 4 S 4 ] cubanes joined
by a shared S-atom-corner, resulting in a l 6 -S atom center in its final geometry
(Fig. 1b). In the dithionite-reduced state (designated as P
N state), the P-cluster is
ligated by six cysteinyl residues: three from the a-subunit (Cysa62, Cysa88, and
Cysa154) and three from the b-subunit (Cysb70, Cysb95, and Cysb153) (Fig. 1b,
top). Each of these six cysteines coordinate with one iron atom, while the Cysa88
and Cysb95 side chains each bridge two Fe centers (Fig. 1b, top). EPR spectroscopy shows that the dithionite-reduced P
N
-cluster is essentially diamagnetic
while Mössbauer spectroscopy demonstrated that it is an all-ferrous iron cluster [27,
28]. Upon treatment with IDS, the P
N -cluster can be oxidized by two electrons to
become the P
ox state. The P
ox cluster displays a S = 3 or 4 integer spin state, which
can be identified by a parallel-mode EPR signal at g * 12 [29]. This signal is often
used as a diagnostic signature of P-cluster given that the P
N cluster is diamagnetic.
This transition from P
N to P
ox also causes the metal cluster to conformationally
rearrange. This rearrangement involves changes in the coordination spheres of two
of the cluster’s iron atoms: While being coordinated initially by the central sulfur
atom in the P
N state, this partner is being replaced by the Oc atom of Serb188 and
the backbone-amide N atom of Cysa88 in the P
ox state (Fig. 1b, bottom) [20, 30].
As a result, one half of the P-cluster, i.e., the half coordinated by the residues in the
b-subunit, assumes a more open form. This dramatic conversion is fully reversible,
and there is evidence that both states, P
N and P
ox , might be physiologically relevant
[8, 31]. Thus, the elaborate structural rearrangement may well serve a unique
purpose in nitrogenase catalysis, possibly related to P-cluster’s role to relay electrons to the M-cluster.
The overall structure of the M-cluster can also be viewed as the product of the
fusion of [MoFe 3 S 3 ] and [Fe 4 S 3 ] cubane-like fragments that are (i) bridged by three
l 2 -sulfide atoms (also known as the “belt sulfur” atoms), and (ii) sharing a unique
l 6 -carbide atom in the center of the M-cluster (Fig. 1c) [18, 32]. In addition, an
organic molecule, R-homocitrate, coordinates the Mo atom in a bidentate fashion
through one hydroxyl- and one carboxyl O atom (Fig. 1c). There is currently no
consensus on the physiologically relevant oxidation states of the M-cluster. In the
dithionite-reduced state, it exhibits a S = 3/2 EPR signal at g = 4.7, 3.7, and 2.0,
which disappears upon enzymatic reduction [4]. Unlike the P-cluster, the M-cluster
is only covalently attached to NifDK by two residues: Hisa442, which binds to the
158
C.-C. Lee et al.
reduced. Remarkably, this process repeats itself multiple times to successively
accumulate sufficient amounts of reducing equivalents for the reduction of N 2 or
other substrates [24–26].
2.2 Structures and Properties of P- and M-Cluster
of Mo-Nitrogenase
Regarded among the most complex metalloclusters in nature, both the P- and the
M-cluster of Mo-nitrogenase can actually be viewed as fused [Fe 4 S 4 ]-like modules.
The [Fe 8 S 7 ] architecture of the P-cluster is essentially two [Fe 4 S 4 ] cubanes joined
by a shared S-atom-corner, resulting in a l 6 -S atom center in its final geometry
(Fig. 1b). In the dithionite-reduced state (designated as P
N state), the P-cluster is
ligated by six cysteinyl residues: three from the a-subunit (Cysa62, Cysa88, and
Cysa154) and three from the b-subunit (Cysb70, Cysb95, and Cysb153) (Fig. 1b,
top). Each of these six cysteines coordinate with one iron atom, while the Cysa88
and Cysb95 side chains each bridge two Fe centers (Fig. 1b, top). EPR spectroscopy shows that the dithionite-reduced P
N
-cluster is essentially diamagnetic
while Mössbauer spectroscopy demonstrated that it is an all-ferrous iron cluster [27,
28]. Upon treatment with IDS, the P
N -cluster can be oxidized by two electrons to
become the P
ox state. The P
ox cluster displays a S = 3 or 4 integer spin state, which
can be identified by a parallel-mode EPR signal at g * 12 [29]. This signal is often
used as a diagnostic signature of P-cluster given that the P
N cluster is diamagnetic.
This transition from P
N to P
ox also causes the metal cluster to conformationally
rearrange. This rearrangement involves changes in the coordination spheres of two
of the cluster’s iron atoms: While being coordinated initially by the central sulfur
atom in the P
N state, this partner is being replaced by the Oc atom of Serb188 and
the backbone-amide N atom of Cysa88 in the P
ox state (Fig. 1b, bottom) [20, 30].
As a result, one half of the P-cluster, i.e., the half coordinated by the residues in the
b-subunit, assumes a more open form. This dramatic conversion is fully reversible,
and there is evidence that both states, P
N and P
ox , might be physiologically relevant
[8, 31]. Thus, the elaborate structural rearrangement may well serve a unique
purpose in nitrogenase catalysis, possibly related to P-cluster’s role to relay electrons to the M-cluster.
The overall structure of the M-cluster can also be viewed as the product of the
fusion of [MoFe 3 S 3 ] and [Fe 4 S 3 ] cubane-like fragments that are (i) bridged by three
l 2 -sulfide atoms (also known as the “belt sulfur” atoms), and (ii) sharing a unique
l 6 -carbide atom in the center of the M-cluster (Fig. 1c) [18, 32]. In addition, an
organic molecule, R-homocitrate, coordinates the Mo atom in a bidentate fashion
through one hydroxyl- and one carboxyl O atom (Fig. 1c). There is currently no
consensus on the physiologically relevant oxidation states of the M-cluster. In the
dithionite-reduced state, it exhibits a S = 3/2 EPR signal at g = 4.7, 3.7, and 2.0,
which disappears upon enzymatic reduction [4]. Unlike the P-cluster, the M-cluster
is only covalently attached to NifDK by two residues: Hisa442, which binds to the
158
C.-C. Lee et al.