Mic 428 Lecture

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Mic 428 - Lecture 10 Nitrogenase and Nitrogen
Fixation
Outline
Nitrogenase and Nitrogen fixation. FeMo
complex. Nitrogenase inhibition by oxygen and the
ways to overcome it. Nitrogenase and alternative
nitrogenases. Functions. Electron flow in
nitrogen fixation.
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Outline continuation
Acetylene reduction as an indirect way of
assaying nitrogenase. Genetics of nitrogen
fixation. Regulation of nitrogen fixation
regulation by oxygen and by ammonia.
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Nitrogen fixation is the process by which
atmospheric nitrogen gas is converted into
ammonia. The reaction can be presented as
follows
Fe
Mo
FeMo-co, where the actual reduction of N2 takes
place
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Structure of FeMo-co
Figure 17-69 Two molecules of FeMo-co are
present per molecule of nitrogenase.
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N N stable triple bond
Its activation is a very energy-demanding process
Six electrons must be transferred and the process
occurs in several steps.
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Nitrogen fixation is inhibited by oxygen since
dinitrogenase reductase is rapidly and
irreversibly inactivated by oxygen.
Nitrogenase is protected from oxygen by several
mechanisms

• Rapid removal of oxygen by respiration.
• Production of slime layers
• Conformational protection

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Alternative nitrogenases
Some nitrogen fixing organisms produce
alternative nitrogenases under Mo limitation.
FeVa co
Function?
Presumably as a backup mechanism.
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Electron flow in nitrogen fixation
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Nitrogenase function
Steps in N2 fixation starting from pyruvate.
Electrons are supplied from dinitrogenase
reductase to dinitrogenase one at a time, and
each electron supplied is associated with the
hydrolysis of 23 ATPs.
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Nitrogenase Function
Hypothetical steps in N2 reduction showing the H2
evolution step, and a summary of nitrogenase
activity.
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Nitrogenase is not entirely specific for nitrogen
since it also reduces cyanide (CN-), acetylene
(HC HC) and several other triply bonded
compounds.
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Acetylene reduction assay for nitrogenase activity
The results show no C2H4 when the experiment
begins (time 0), but increasing production of
C2H4 as the assay proceeds. Note how as C2H4 is
produced, C2H2 is consumed.
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Genetics and Regulation of Nitrogen Fixation
Genetics
The nif regulon
It contains approximately 20 genes arranged in
several transcriptional units.
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The nif regulon in Klebsiella pneumoniae, the
best-studied nitrogen-fixing organism
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Nitrogenase
Product of gene nifD
Product of gene nifK
Product of gene nifH
FeMo-co, where the actual reduction of N2 takes
place
several genes
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Regulation
Nitrogen fixation blocked by oxygen and by fixed
nitrogen (ammonia, nitrate and some aminoacids).
Transcription of nif structural genes is
repressed by NifL protein that functions shutting
down transcription of nif genes in the presence
of oxygen.
Ammonia represses nitrogen fixation through a
protein called NtrC that is active when ammonia
is active and promotes transcription of nifA.