Bacteria Metabolism Flashcards
1
Q
What is the size of global nitrogen fixation?
A
3x10^11 kg/year
- 50% = biological N fixation (prokaryotes have 50% more flux through N fixation than natural sources)
- 50% = industrial synthesis (e.g. Haber Bosch process, inefficient, low capture of N20 which leaks into atmosphere
- 1% = lightning
2
Q
What organisms fix nitrogen (+ 2 examples)
A
- Either eubacteria or archaea
- Bacteria give plants fixed N, plants give fixed C
- Proteobacteria (e.g. Rhizobium) + leguminous plants (nodules are infected when Rhizobia infect root hairs of compatible leguminous plants
- Actinorhizal plants + actinomycetes (gram +ve bacteria of genus Frankia)
3
Q
Why is dinitrogen chemically inert? (thermodynamic)
A
- Triple N bond = v strong + endergonic
- N2 + H2 → N2H2 ΔH = +50.9kcal/mol
- 2nd step = -27.2 3rd = -45.6
4
Q
Why is dinitrogen chemically inert? (kinetic)
A
- N2 = non polar
- Has tightly bound sigma + pi e-s so poor ligand
5
Q
Nitrogenase overall reaction
A
N2 + 8H+ + 8e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi
6
Q
Features of nitrogenase (1-6)
A
- Reaction is v. energetically costly (2.5x CO2 fixation in Calvin cycle). Exergonic w/o ATP hydrolysis
- e- donor = either ferredoxin or flavodoxin (carry e-s at more reducing potential than NADH/NADPH
- Has low potential redox centres, needs to be protected from inactivation by O2
- H2 production is obligate part of reaction
- Slow turnover (5s-1)
- Other molecules w/ multiple bonds can be reduced (acetylene → ethene, used as probe)
7
Q
MoFe protein
A
- Heterotetramer. Composed of a + B subunits (a2B2), form pseudo-symmetry
- Has FeMo cofactor in a subunit
- Has P cluster at centre of subunit-subunit interface
- Reduced P cluster = ligand w. 6 lys residues, central S1 sulfide is coordinated w/ 6Fe
8
Q
Fe protein + structure
A
- Homodimer, binds 4Fe-4S cluster at interface btw 2 subunits
- Open conformation (x bound MoFe, ADP bound)
- Closed conformation (complexed w/ MoFe. complete catalytic site)
- Closed conformation brings 2 catalytic residues (lys10 + asp129) from other subunits close
9
Q
Fe protein cycle steps (1-7)
A
- Fe protein forms a complex w/ MoFe proteinATP
- e- transfer from Fe to MoFe is coupled to ATP hydrolysis
- Phosphate is release, rate limiting step
- ADP-bound, oxidised Fe protein is released
- Fe protein re-reduced by ferredoxin/flavodoxin
- ATP exchanged for ADP in Fe protein
- Cycle repeated x8 as need 8e-
10
Q
Electron transfer distance (+AlF-.ADP)
A
- AlF4.ADP = TS analogue of nucleotide hydrolysis
- e- transfer ↓ w/ edge-edge separation btw active redox species. 14A = max separation
- Distances: 13A. 14A
4Fe-4S → P cluster → FeMoco - So, know it’s this and not 4Fe-4S → FeMoco
11
Q
Complex btw Fe + MoFe
Fe protein cycle
A
- Tightest fit w. ADP.AlF4-
- Loose asymmetric fit in ADP-loaded complex
- Distance btw Fe-protein iron-sulfur cluster + P cluster from 18→13A (open→closed)
- 4Fe-4S cluster is pushed to apex of Fe protein in closed, ↓ e- transfer distance to P cluster
12
Q
Complex btw Fe + MoFe mechanism
Fe protein cycle
A
- MoFe protein stab. closed state of Fe protein
- This change to closed brings 4Fe-4S within e- transfer distance of P cluster
- ATP hydrolysis also occurs
- ADP state = loosening of interactions btw Fe + MoFe
13
Q
Models for use of Fe protein cycle
A
- Super reducing e- model
- ATP hydrolysis ↓ reduction potential of e-s delivered by Fe protein
- Change in conf. changes environment and reduction potential by -200mV
- But low chemical reductants x support dinitrogen reduction - Deficit spending model of e- flow
- ATP bound form of the Fe protein drives conf. change in the MoFe that drives e- from P cluster to FeMoco
- This e- is replaced by e- from Fe protein to P cluster
- For = P cluster is already fully reduced so x initially receive e- from protein crystal
- Against = no conf. change on Fe protein binding in MoFe crystal structure
14
Q
Catalysis at FeMoco
A
- Structure of FemoCo adducts suggest the cofactor cage rearranges during catalysis
- Turnover w/ inhibitor CO → CO replaces surface ion that bridges Fe2+6
15
Q
Lowe Thornley Kinetic model
A
- Steady state, freeze quench + stopped flow kinetics carried out by Lowe and Thronley
- At each stage, 1e- + 1H+ is added, so cofactor have same charge
- MoFe goes from Eo to E8 during N2 fixation
- 4e- need to be transferred before adding N2, although can react w/ E3
16
Q
Glutamate dehydrogenase
A
NH4+ + a-ketoglut. + NADPH + H+ → glutamate + NADP+ + H20
17
Q
Glutamine synthetase
A
NH4+ + glutamate + ATP → glutamine + ADP + Pi + H+
18
Q
GOGAT/ glutamine synthase
A
a-ketoglut + glutamine + NADPH + H+ → 2glutamate + NADP+
19
Q
High NH4+ constitutive pathway
A
2NH4+ + a-kg → 2 glutamate → 2 glutamine
- 1st step = glutamate dehydrogenase (+ a-kg + 4e-)
- 2nd step = glutamine synthase, (-a-kg, -2e-)
20
Q
Low NH4+ inducible pathway
A
NH4+ + glutamate → glutamine → 2 glutamate
- 1st step = glutamine synthetase
- 2nd = glutamine synthase
21
Q
NtrBC
A
- Controls expression of glutamine synthetase
- Ntr system senses glutamine x ammonia as better to know N donor
- Also responds to a-ketoglutarate
22
Q
PII proteins
A
- Trimeric proteins
- Split into 3: glnB, GlnK + nifI
- Modified by UTase/UR (encoded by GlnD)
- PII-UMP de-adenylates + activates GS by ATase
- PII adenylates + inactivates GS
- ↓ NH4+ = UTase =PII-UMP
= active GS
23
Q
At/AR
A
?
24
Q
Nitrate reductase
Nas operon
A
- NO3- + 2H+ + 2e- → NO2- + H20
- If x fix ↓ glutamine w/ GS, protein overcome by activating genes for nitrate reductase
- Nitrate x used as N source when NH4+ ↑.
- Means cell x have to provide low potential reductants or make additional E
- nas operon (σ54)
25
Nitrite reductase
| Nas operon
- NO2- + 8H+ + 6e- → NH4+ + 2H20
- In plants = plasticity E, H+ dependent nitrite transporters transport nitrite into chloroplast to sustain nitrate assimilation
26
NifA
- Lowest affintiy promoter?
- σ54 promoter + needs EBP = NifA
- Transcription of NifLA needs NtrC-P + σ54
- Oxidised NifL binds NifA + prevents it activating nif genes (oxidation sated = controlled by ETC)
- Prevents O2 damaging nitrogenase
27
Hierarchy of affinity for NtrC
```
Highest = glnA
Mid = nas
Lowest = nifA
```
28
Oxygen conundrum (Nitrogen fixation)
/
Protection of nitrogenase against O2 - leghemoglobin
- E generation in bacteriods needs reduced O2 like mammalian ETC
- BUT, need to keep [O2] ↓ for nitrogenase
- Leghemoglobin = ↑ conc. + has ↑ affinity for O2 (Kd - 20nm). Maintains ↓ o2
- Assists O2 moving to cyt c oxidase which has ↑ affinity for O2
- So, keeps high flux through ETC
29
Rhizobia reduction of ferredoxin
- x use well known pathway of reduction ferredoxin like PSI
- Instead use NADH as e- donor
- Normally x reducing enough to reduce ferredoxin
- But, 2e-s follow different paths, like complex III
- Reducing power ion NADH is unequally divided btw 2e-s
- More reducing e- = ↓ enough reduction potential to reduce ferredoxin
- Less reducing route reduces ubiquinone
30
FixLJ
- Directly senses O2
- Fix J = DNA response regulator
- Fix L = sensor kinase
- Fix L directly senses O2, binds haem group in the sensor domain + inactivates its kinase activity
31
Protection of nitrogenase against O2 - heterocyst
- Cyanobacteria
- Challenge = O2 = main product of photosynthesis
- Solve issue of O2 by segregating N-fixing E in specialised heterocyst
- Heterocyst lacks PSII so x make O2 + = physical barrier
32
P cluster
- Part of MoFe protein
- Thought to be 2 symmetric [Fe4S4] cubanes share a central S so [8Fe-7S]
- Thought to mediate e- transfer btw the Fe protein + substrate reduction site FeMoco
- Evidence = X-ray crystal structures of two different stable Fe protein-MoFe protein complexes place the P-cluster between the Fe protein [4Fe-4S] cluster and FeMo-cofactor
33
Sources of nitrogen
- aa are best as once glutamine/glutamate are synthesised, no further costs
- N2 is worst as is very stable, E is slow, ↑ cost to assimilate
34
NtrB/C
- Cotranscribed in glnA-ntrBC operon
- NtrC = σ54 transcriptional activator. Has AAA+ ATPase activity that melts DNA at the promoter
- ↑ N, PII → NtrB phosphorylates + deactivates NtrC
- ↓ N, PII-UMP, NtrC-P stays
35
NtcA
- Analogous to NtrC
- Controls ammonia repression of nitrate assimilation in cyanobacteria
- Activated by PipX
- When ↓ N, NtcA-PipX x PipX-PII
36
AmtB
- Ammonia transporter
- Activation requires NtrC-P
- ↑ N, GlnK/PII x UMP is sequestered to membrane by amtB, prevents NH4+ entering
- ↓ N, GlnK-UMP, x bind AmtB, NH4+ enters
37
Nas genes
- Regulator proteins = 3 types: NtcB regulator of cyanobacteria, 2 component regulatory system NasST or NasR regulator protein of heterotrophic bacteria
- E.g. NasR
- NasR binds nas operon mRNA + prevents hairpin formation that causes termination of transcription
- ↑ N, nasFED-CBA operon is repressed by ↓ TasE + ↑ activation of NasF promoter
