Roger Thorneley in 1984, and later embellished by Hoffman and others [9, 81, 82].
While it was chiefly invented to describe the reduction of N 2 , it still provides a
framework for the catalytic mechanism of nitrogenase in general and is also frequently used to explain the reduction of other alternative substrates by this enzyme
[4]. The model defines eight discrete stages of the catalytic cycle of NifDK, where
each stage represents one of the eight electron- and proton-transfer steps that are
proposed to fuel the conversion of one molecule of N 2 to two NH 3 molecules, while
forming one molecule of H 2 as the obligatory side product (Fig. 6a). The individual
stages are denoted as E n , where n represents the number of electrons and protons
accumulated on one ab-dimer of NifDK—either associated with the P-cluster or the
M-cluster. The first state is E 0 , which is the dithionite-reduced, as isolated NifDK. It
has an overall spin state of S = 3/2, thus indicating an odd number of unpaired
electrons. Following this logic, it can be deduced that all even-numbered states (i.e.
E n where n = 2, 4, 6, etc.) will have an odd number of unpaired electrons as well
and are hence paramagnetic. In contrast, the odd-numbered E n states (i.e. E n where
n = 1, 3, 5, etc.) will have an even number of unpaired electrons and should either
be diamagnetic or possess integer spin states (S = 1, 2, 3, etc.) which are often
undetectable by EPR. Therefore, preliminary information regarding the spin state of
any nitrogenase reaction intermediate species can already be very helpful in
determining whether an even or odd number of electrons and protons have been
added into the system [81, 82]. This feature was often being taken advantage of
when a purported intermediate species is observed of which the catalytic stage it
arises from is unclear.
Upon receiving electrons from the Fe protein, the E 0 state is reduced stepwise to
a series of turnover states. It is believed that in the absence of suitable substrates,
the states E 2 to E 4 are oxidized back to the states E 0 to E 2 . This oxidation process, in
which two electrons are removed, is coupled to the formation of H 2 (Fig. 6a) [83,
84]. By only cycling through the states E 0 to E 4 , Mo-nitrogenase effectively
becomes an ATP-dependent hydrogenase in the absence of proper substrates.
Regarding the reduction of N 2 , it is now commonly believed that the binding of N 2
occurs at the E 4 state, which proposedly occurs through a reductive elimination
mechanism (Fig. 6) [81, 82, 85–87]. After that, NifDK can undergo five more
successive proton/electron-transfer steps to cleave the NN triple bond and
eventually yield two molecules of NH 3 (Fig. 6a). Beginning with the states E 4 to
E 6 , two distinct reaction pathways were proposed for the reduction of the activated
N 2 to the two NH 3 products [4, 82]. In the so-called distal pathway, which was
described in the original LT scheme, one N atom (namely the distal N) is first
reduced all the way to NH 3 and released before the reduction of the second N
begins. A later proposal by Seefeldt and Hoffmann, known as the alternating
pathway, describes the alternating reduction on the two N atoms so that both NH 3
are produced and released in the last two states successively (Fig. 6a) [9, 82].
Interestingly, these two proposals predict completely different sets of reaction
intermediates at the states E 4 , E 5 , and E 6 , while still converging at a common E 7
state (Fig. 6a).
Assembly and Function of Nitrogenase
171
While it was chiefly invented to describe the reduction of N 2 , it still provides a
framework for the catalytic mechanism of nitrogenase in general and is also frequently used to explain the reduction of other alternative substrates by this enzyme
[4]. The model defines eight discrete stages of the catalytic cycle of NifDK, where
each stage represents one of the eight electron- and proton-transfer steps that are
proposed to fuel the conversion of one molecule of N 2 to two NH 3 molecules, while
forming one molecule of H 2 as the obligatory side product (Fig. 6a). The individual
stages are denoted as E n , where n represents the number of electrons and protons
accumulated on one ab-dimer of NifDK—either associated with the P-cluster or the
M-cluster. The first state is E 0 , which is the dithionite-reduced, as isolated NifDK. It
has an overall spin state of S = 3/2, thus indicating an odd number of unpaired
electrons. Following this logic, it can be deduced that all even-numbered states (i.e.
E n where n = 2, 4, 6, etc.) will have an odd number of unpaired electrons as well
and are hence paramagnetic. In contrast, the odd-numbered E n states (i.e. E n where
n = 1, 3, 5, etc.) will have an even number of unpaired electrons and should either
be diamagnetic or possess integer spin states (S = 1, 2, 3, etc.) which are often
undetectable by EPR. Therefore, preliminary information regarding the spin state of
any nitrogenase reaction intermediate species can already be very helpful in
determining whether an even or odd number of electrons and protons have been
added into the system [81, 82]. This feature was often being taken advantage of
when a purported intermediate species is observed of which the catalytic stage it
arises from is unclear.
Upon receiving electrons from the Fe protein, the E 0 state is reduced stepwise to
a series of turnover states. It is believed that in the absence of suitable substrates,
the states E 2 to E 4 are oxidized back to the states E 0 to E 2 . This oxidation process, in
which two electrons are removed, is coupled to the formation of H 2 (Fig. 6a) [83,
84]. By only cycling through the states E 0 to E 4 , Mo-nitrogenase effectively
becomes an ATP-dependent hydrogenase in the absence of proper substrates.
Regarding the reduction of N 2 , it is now commonly believed that the binding of N 2
occurs at the E 4 state, which proposedly occurs through a reductive elimination
mechanism (Fig. 6) [81, 82, 85–87]. After that, NifDK can undergo five more
successive proton/electron-transfer steps to cleave the NN triple bond and
eventually yield two molecules of NH 3 (Fig. 6a). Beginning with the states E 4 to
E 6 , two distinct reaction pathways were proposed for the reduction of the activated
N 2 to the two NH 3 products [4, 82]. In the so-called distal pathway, which was
described in the original LT scheme, one N atom (namely the distal N) is first
reduced all the way to NH 3 and released before the reduction of the second N
begins. A later proposal by Seefeldt and Hoffmann, known as the alternating
pathway, describes the alternating reduction on the two N atoms so that both NH 3
are produced and released in the last two states successively (Fig. 6a) [9, 82].
Interestingly, these two proposals predict completely different sets of reaction
intermediates at the states E 4 , E 5 , and E 6 , while still converging at a common E 7
state (Fig. 6a).
Assembly and Function of Nitrogenase
171