15
Tezcan 2020). The metal centers play key roles in nitrogenase function. In the case
of the molybdenum (Mo) enzyme these are the MoFe 7 S 9 in the cofactor containing
homocitrate termed FeMoCo and a P cluster (Fe 8 S 7 ) (Cohen 2014; Howard and
Rees 1996) (Fig. 2.1). The Fe protein dimer binds a Fe 4 S 4 between cysteine residues
of the subunits (Cohen 2014) and each subunit binds one ATP at Walker binding
motifs (Rutledge and Tezcan 2020). The Fe 4 S 4 is exposed near the surface of the
dimer in a way that likely facilitates electron transfer to the P cluster of the MoFe
protein when the two proteins interact (Benton et al. 2002). The redox potential of
the Fe 4 S 4 is affected by nucleotide binding which is probably important in coupling
electron transfer to ATP hydrolysis.
2.3 The Biological Nitrogen Fixation Reaction
The biological reduction of N 2 with nitrogenase is a complex multi-step reduction
reaction. The nitrogenase reaction has been of great interest for decades because of
its importance to agriculture but also because of its complex biochemistry (Fisher
and Newton 2002). Much of what is known about the nitrogenase reaction is from
work in vitro as well as in vivo, so many details are understood, but the specifics of
the reaction in vivo remain poorly elucidated. The triple-bonded N 2 molecule is very
stable, and has a high activation energy to break. The first model, involving 7 steps
of electron transfers (E 1 -E 7 ) was proposed by (Thorneley and Lowe 1985) and has
been modified more recently (reviewed in Seefeldt et al. 2020).
Electrons are supplied to dinitrogenase reductase from a low potential biological
molecule, either ferredoxin or flavodoxin, which pass electrons to the Fe protein
(Fig. 2.3). In overview, ATP is bound to the reduced Fe protein, the Fe protein docks
with the MoFe protein, electrons are transferred, ATP is hydrolysed, and the proteins undock (Fisher and Newton 2002). The binding and release of the Fe protein
is called the Fe protein cycle (Seefeldt et al. 2020). After docking, electrons are first
transferred from the P cluster to the metal cofactor MoFe (or V, or Fe) center, and
then replaced from the Fe-S center of the Fe protein dimer. The reaction at the site
of reduction of N 2 and H 2 is still somewhat unclear but involves multiple electron
transfers and reductive elimination/oxidative addition (re/oa) at the E4 state where
4H are bonded as hydrides. This state can resolve by reductively eliminating H 2 or
oxidatively adding H. Currently it is thought that this may involve a central carbide
in FeMoCo (Rutledge and Tezcan 2020; Seefeldt et al. 2020; Siegbahn 2019;
Spatzal et al. 2011). The key step is E4 where N 2 displaces H 2 in the active site.
Many details of how the electrons are transferred, when and how ATP is hydrolysed
and how H 2 is evolved are still controversial (Rutledge and Tezcan 2020). The Fe
protein interacts with the MoFe protein with the FeS center close to the P cluster of
the MoFe protein (Rutledge and Tezcan 2020). Both the conformation of the Fe
protein and the geometry of binding to the MoFe protein are affected by nucleotide
(ATP) binding. Electrons are transferred one at a time from the Fe protein to the Mo
2.3 The Biological Nitrogen Fixation Reaction
Tezcan 2020). The metal centers play key roles in nitrogenase function. In the case
of the molybdenum (Mo) enzyme these are the MoFe 7 S 9 in the cofactor containing
homocitrate termed FeMoCo and a P cluster (Fe 8 S 7 ) (Cohen 2014; Howard and
Rees 1996) (Fig. 2.1). The Fe protein dimer binds a Fe 4 S 4 between cysteine residues
of the subunits (Cohen 2014) and each subunit binds one ATP at Walker binding
motifs (Rutledge and Tezcan 2020). The Fe 4 S 4 is exposed near the surface of the
dimer in a way that likely facilitates electron transfer to the P cluster of the MoFe
protein when the two proteins interact (Benton et al. 2002). The redox potential of
the Fe 4 S 4 is affected by nucleotide binding which is probably important in coupling
electron transfer to ATP hydrolysis.
2.3 The Biological Nitrogen Fixation Reaction
The biological reduction of N 2 with nitrogenase is a complex multi-step reduction
reaction. The nitrogenase reaction has been of great interest for decades because of
its importance to agriculture but also because of its complex biochemistry (Fisher
and Newton 2002). Much of what is known about the nitrogenase reaction is from
work in vitro as well as in vivo, so many details are understood, but the specifics of
the reaction in vivo remain poorly elucidated. The triple-bonded N 2 molecule is very
stable, and has a high activation energy to break. The first model, involving 7 steps
of electron transfers (E 1 -E 7 ) was proposed by (Thorneley and Lowe 1985) and has
been modified more recently (reviewed in Seefeldt et al. 2020).
Electrons are supplied to dinitrogenase reductase from a low potential biological
molecule, either ferredoxin or flavodoxin, which pass electrons to the Fe protein
(Fig. 2.3). In overview, ATP is bound to the reduced Fe protein, the Fe protein docks
with the MoFe protein, electrons are transferred, ATP is hydrolysed, and the proteins undock (Fisher and Newton 2002). The binding and release of the Fe protein
is called the Fe protein cycle (Seefeldt et al. 2020). After docking, electrons are first
transferred from the P cluster to the metal cofactor MoFe (or V, or Fe) center, and
then replaced from the Fe-S center of the Fe protein dimer. The reaction at the site
of reduction of N 2 and H 2 is still somewhat unclear but involves multiple electron
transfers and reductive elimination/oxidative addition (re/oa) at the E4 state where
4H are bonded as hydrides. This state can resolve by reductively eliminating H 2 or
oxidatively adding H. Currently it is thought that this may involve a central carbide
in FeMoCo (Rutledge and Tezcan 2020; Seefeldt et al. 2020; Siegbahn 2019;
Spatzal et al. 2011). The key step is E4 where N 2 displaces H 2 in the active site.
Many details of how the electrons are transferred, when and how ATP is hydrolysed
and how H 2 is evolved are still controversial (Rutledge and Tezcan 2020). The Fe
protein interacts with the MoFe protein with the FeS center close to the P cluster of
the MoFe protein (Rutledge and Tezcan 2020). Both the conformation of the Fe
protein and the geometry of binding to the MoFe protein are affected by nucleotide
(ATP) binding. Electrons are transferred one at a time from the Fe protein to the Mo
2.3 The Biological Nitrogen Fixation Reaction