Metabolism VII: Diazotrophy (read extra) rewatch Flashcards
overview of diazotrophy
- diazotrophy, “nourishment from molecular nitrogen”. Use of N2 as a nitrogen source.
- much more specialist than using e.g. NH4+ or NO3
- etc and VERY expensive!
- free-living diazotrophs are just that: live freely in soils etc.
- plant-associated diazotrophs are found in symbiosis with the Viridiplantae – these include both root-nodule and non-root-nodule types.
spit out ammonia
root-nodule diazotrophs
- members of the family Rhizobiaceae are found inside of plant root nodules (but sometimes have a free-living life
phase too) in the Fabales. - members of the genus Frankia are found inside plant root nodules and root hairs of various different plant taxa in the Fagales and few members of Rosales.
Phaseolus coccineus (runner beans) showing root nodules grow very fast due to amount of nitrogen they can get.
non-root nodule diazotrophs
- a small group of Nostoc spp. from the former they live inside plant stem they.
Cyanobacteria” found above ground in Gunnera spp. - a small group of Anabaena spp. found in Azolla spp. (floating fern)
- a small group of Nostoc spp. found in the coralloid roots of various Cycadopsida and in the tissues of various Marchantiophyta.
Cycas subgen. Panzhihuanensis sect. Asiorientales revoluta
showing coralloid root they are a sort of root nodule for nostoc hence they sit on soil
abiotic equivalent reactions
Haber-Bosch process is used commercially to reduce molecular nitrogen
to ammonia:
N2 + 3H2 → 2NH3 ΔG° = -26.6 kJ/mol NH3 produced
* in spite of the reaction releasing energy to the universe, it only works at 98.7 atm, 450 ℃ using iron-metal oxide catalysts.
* why? The activation energy (EA) is so high (c.+700 kJ/mol NH3 produced), so it doesn’t actually occur spontaneously!
In life:
N2 + 6H+ → 2NH3 ΔG° = -33.0 kJ/mol NH3 produced
* EA cost (lower in Life because nitrogenase mechanism is different to ruthenium catalyst mechanism) is overcome by coupling the reaction to the oxidation of a reduced electron carrier and hydrolysis of ATP (actual reaction varies as several nitrogenase enzymes) to ‘pay’ for EA – one
example:
4Fd(red) + 16ATP → 4Fd(ox) + 16ADP + 16Pi
ΔG° = -201.2 kJ/mol ATP hydrolysed
very expensive
GIve some examples
first one is most important
- Azotobacter vinelandii and Azotobacter chroococcum
[N.L. neut. n. azotum, nitrogen; N.L. masc. n. bacter, rod; N.L. masc. n. Azotobacter, a nitrogen rod; N.L. gen. n. vinelandii, of Vineland, New Jersey; Gr. masc. n. χρώς (khrṓs),
colour; N.L. masc. n. coccus, a seed; N.L. neut. n. chroococcum, coloured balls]
[Pseudomonadaceae < Pseudomonadales < Gammaproteobacteria < Pseudomonadota < Pseudomonadati < Bacteria] - Azonexus caeni
[Azonexaceae < Rhodocyclales < Betaproteobacteria < Pseudomonadota < Pseudomonadati < Bacteria]
[N.L. neut. n. azotum, nitrogen; L. masc. n. nexus, a coil [poetic Latin only]; N.L. masc. n. Azonexus, a nitrogen coil; L. gen. n. caeni, of dirt, of filth] - Klebsiella pneumoniae subsp. pneumoniae
[N.L. fem. dim. n. Klebsiella, named for Edwin Klebs (1834-1913); N.L. gen. n. pneumoniae, of pneumonia]
[Enterobacteriaceae < Enterobacterales < Gammaproteobacteria < Pseudomonadota < Pseudomonadati < Bacteria] - Bradyrhizobium japonicum
[Gr. masc. adj. βρᾰδῠ́ς (bradús), slow; Gr. fem. n. ῥῐ́ζᾰ (rhíza), a root; Gr. masc. n. βῐ́ος (bíos), life; N.L. neut. n. Bradyrhizobium, slowgrowing root life-form; N.L. neut. adj. japonicum, pertaining to Japan]
[Nitrobacteraceae < Hyphomicrobiales < Alphaproteobacteria < Pseudomonadota < Pseudomonadati < Bacteria] - Methylococcus capsulatus
[N.L. neut. n. methylum, the methyl group; N.L. masc. n. coccus, a seed; N.L. masc. n. Methylococcus, a methyl-using ball; N.L. masc. adj.
capsulatus, encapsulated]
[Methylococcaceae < Methylococcales < Gammaproteobacteria < Pseudomonadota < Pseudomonadati < Bacteria] - Nostoc commune
[Eng. n. nostril, orifice in the nose; Germ. neut. n. Nasenloch, orifice in the nose; N.L. neut. n. Nostoc, something from the nose, snot; L. neut. adj.
commune, commonplace]
[Nostocaceae < Nostocales < Cyanophyceae < Cyanobacterota < Bacillati < Bacteria] - Heliobacterium chlorum
[Gr. masc. n. ἥλῐος (hḗlios), sun; Gr. neut. n. βακτήριον (baktḗrion), small staff; N.L. neut. n. Heliobacterium, sun bacterium; Gr. neut. adj. χλωρόν (khlōrón), the
verdant green of new spring growth; N.L. neut. adj. chlorum, the verdant green of new spring growth]
[Heliobacteraceae < Eubacterales < Clostridia < Bacillota < Bacillati < Bacteria] - Paenibacillus polymyxa
[L. adv. paene, almost, nearly; L. masc. n. bacillus, a rod and Bacillus, the name of a genus; N.L. masc. n. Paenibacillus, almost a Bacillus; Gr. masc. adj. πολῠ́ς
(polús), many, a lot; Gr. fem. n, μῠ́ξᾰ (múxa), mucus, slime; N.L. masc. n. polymyxa, producing a lot of slime]
[Paenibacillaceae < Caryophanales < Bacilli < Bacillota < Bacillati < Bacteria] - Methanococcus maripaludis (archaea)
[N.L. neut. n. methanum, methane; N.L. masc. n. coccus, a seed; N.L. masc. n. Methanococcus, a methanogenic ball; L.
gen. n. maris, of a sea; L. gen. n. paludis, of a marsh; N.L. gen. n. maripaludis, of a sea marsh]
[Methanococcaceae < Methanococcales < Methanococci < Methanobacteriota < Methanobacteriati < Archaea]
nitrogenase enzymes
- ultimately convert molecular nitrogen (N2) into
ammonia/ammonium (NH3/NH4+). - literature is a bit confusing as phenotypes and proteins
purified pre-sequencing don’t fully match up with what
we know now! - THREE types of nitrogenase.
- nitrogen uptake from ammonia onwards is ‘as
normal’ (GS/GOGAT pathways – not part of this
module!). - precise mechanism is complex e.g. Lowe-Thorneley
model, Janus intermediates etc – do not try and learn
this for this module! - costly: whilst N2 reduction has a negative Gibbs energy,
it has a huge activation energy, so much ATP is needed! - nitrogenases get inhibited by molecular oxygen (O2).
- quantified in vitro based on reduction of acetylene
(ethyne) to ethylene (ethene) and/or ethane or
sometimes based on molecular hydrogen (H2) emission.
nitrogenase (Mo-dependent) EC 1.18.6.1
requires Mo and Fe as bound metals.
* Mg2+ required for activity.
* ferredoxin (Fd) cofactor.
N2 + 8H+ + 4Fd(red) + 16ATP + 16H2O → 2NH3 + H2 + Fd(ox) + 16ADP + 16Pi
* NifD and NifK subunits (c. 30 kDa each, Fe-bound), forming
Component I.
* two NifH subunits (c. 60 kDa each, MoFe-bound), forming
Component II.
* NifY not part of protein but needed for it to properly mature.
* can reduce acetylene to ethylene.
* cannot reduce carbon monoxide – this is an inhibitor.
* found in Anabaena, Nostoc (Cyanophyceae) and Azotobacter, Beggiatoa, Klebsiella (Gammaproteobacteria).
nitrogenase (V-dependent) EC 1.18.6.2
- requires V and Fe as bound metals. (vanadium is quite rare in soils)
- Mg2+ required for activity.
- ferredoxin (Fd) cofactor.
N2 + 12H+ + 12Fd(red) + 40ATP + 40H2O → 2NH3 + 3H2 + 12Fd(ox) + 40ADP + 40Pi - VnfD and VnfK subunits (c. 55 kDa each, Fe-bound), forming Component I.
- two VnfG subunits (c. 60 kDa each, VFe-bound), forming
Component II. - VnfY not part of protein but needed for it to properly mature.
- can reduce acetylene to ethylene.
- can reduce carbon monoxide to ethane, ethene and propane – not very specific reaction!
- found in Anabaena spp. and Azotobacter spp. as before.
nitrogenase (flavodoxin) EC 1.19.6.1
- requires Fe as a bound metal.
- Mg2+ required for activity.
- flavodoxin (NifF) cofactor.
N2 + 2H+ + 4NifF(red) + 16ATP + 16H2O → 2NH4
+ + H2 + 4NifF(ox) + 16ADP + 16Pi - NifD and NifK subunits (c. 55 kDa each, Fe-bound).
- NifF flavodoxin
NifJ pyruvate:flavodoxin oxidoreductase reboots NifF:
NifF(ox) + pyruvate + CoA + H+ → acetyl-CoA + CO2 + NifF(red) - NifY not part of protein but needed for it to properly mature.
- can reduce acetylene to ethylene and slowly on to ethane.
- cannot reduce carbon monoxide – this is an inhibitor.
- found in Azotobacter spp. (as before) and Rhodobacter capsulatus (Alphaproteobacteria).
used in starvation when there isn’t vanadium or the other one
?nitrogenase (alternative) no EC yet
no one knows really
- requires only Fe as bound metal.
- Mg2+ required for activity.
- ferredoxin (Fd) cofactor.
N2 + 8H+ + 4Fd(red) + 16ATP + 16H2O → 2NH3 + H2 + 4Fd(ox) + 16ADP + 16Pi - AnfD and AnfK subunits (Fe-bound), forming Component I.
- two AnfG subunits (Fe-bound), forming Component II.
(A for alternative) - seems to be a variation of VnfDKG – jury is out on whether or not they are truly separate enzymes.
regulation: Nif-based example
- O2 inhibits the enzyme itself.
- normally NifA (regulatory protein) blocks nifDHK expression but is turned off by low O2and low NH4+.
- if O2 is high, causes expression of fumarate and nitrate
reduction regulator (Fnr) which causes oxidation of NifL protein (from FADH2 cofactor to FAD+) – the latter form cannot inhibit NifA, so NifA blocks nifDHK expression. This is one mechanism for coping with O2! - if NH4+
is high, NifR is inhibited, which allows nifA expression, in
turn blocking nifDHK. - if NH4+
is low, Fnr(ox) is blocked, which stops it blocking NifA and
thus nifDHK are expressed.
dealing with O2
read Parker paper
- coat cells/colonies in copious extracellular polymeric
substance (EPS) and/or capsular polymeric
substance (CPS) capsules, which slow O2 diffusion
into cells – usually coupled with a switch from an aa3-
type terminal cyt c oxidase for respiration to a cbb3-
type, which has a much higher O2 affinity. - for root-nodule-based examples, they can induce the
host plant to express leghemoglobin (keeps hold of oxygen so stops it from going near the cells) (one of the
phytoglobin family) which binds O2. Sort-of resembles
myoglobin from the Metazoa. - in the Cyanobacterota with filamentous habits, they
only use nitrogenases in specialist cells called
heterocysts in which no respiration takes place – these
cells have thick CPS capsules to slow O2 diffusion. ATP
is sent into heterocysts from local cells and NH4+ is sent
back out to them.
Read into this
root nodules of Glycine max (L.) Merr. of the Fabales
showing red pigmentation from leghemoglobin
Stem glands of Gunnera tinctoria (Molina) Mirbel.
(arrowed) found at base of stem but also at leaf-bases.
Section through Gunnera tinctoria (Molina) Mirbel. stem glands showing infiltration by Nostoc spp.
anabaena
Heterocyst and normal cell of Anabaena sp. PCC 7120
showing heterocyst polysaccharide (Hep),
heterocyst glycolipid layer (Hgl) and cyanophycin
granule (CG) plus honeycomb-like intracellular
membranes.
cyanophycin = an arginineaspartic acid polymer used as
an N-store.
no thylakoid of carboxysome stores cyanophycin in the poles as this is where it’s needed.
Anabaena solitaria showing normal vegetative cells (dark green uniform cells), an akinete (large cylindrical body with internal bodies) and a heterocyst (pale-green round cell).
akinete – a thick-walled, metabolically dormant cell that has no motile abilities. Resists desiccation and low temperatures and can act as a storage-state for 10-50 years. We will meet them again in L21 in May.
what to read
explain diff and similarity between enzymes look up abundance (occurrence) of vanadium, ion ins soils, water. Bioavailable/bioaccessible.
learn diff strategies of oxygen (might be in exam)?? rewatch
look at review articles after 2007
