Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Paper 2 > Bacteria Metabolism > Flashcards

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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Why is dinitrogen chemically inert? (kinetic)

A
  • N2 = non polar

- Has tightly bound sigma + pi e-s so poor ligand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Nitrogenase overall reaction

A

N2 + 8H+ + 8e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Features of nitrogenase (1-6)

A
  1. Reaction is v. energetically costly (2.5x CO2 fixation in Calvin cycle). Exergonic w/o ATP hydrolysis
  2. e- donor = either ferredoxin or flavodoxin (carry e-s at more reducing potential than NADH/NADPH
  3. Has low potential redox centres, needs to be protected from inactivation by O2
  4. H2 production is obligate part of reaction
  5. Slow turnover (5s-1)
  6. Other molecules w/ multiple bonds can be reduced (acetylene → ethene, used as probe)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Fe protein cycle steps (1-7)

A
  1. Fe protein forms a complex w/ MoFe proteinATP
  2. e- transfer from Fe to MoFe is coupled to ATP hydrolysis
  3. Phosphate is release, rate limiting step
  4. ADP-bound, oxidised Fe protein is released
  5. Fe protein re-reduced by ferredoxin/flavodoxin
  6. ATP exchanged for ADP in Fe protein
  7. Cycle repeated x8 as need 8e-
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Models for use of Fe protein cycle

A
  1. 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
  2. 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
Q

Nitrite reductase

Nas operon

A
  • NO2- + 8H+ + 6e- → NH4+ + 2H20
  • In plants = plasticity E, H+ dependent nitrite transporters transport nitrite into chloroplast to sustain nitrate assimilation
26
Q

NifA

A
  • 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
Q

Hierarchy of affinity for NtrC

A 
Highest = glnA
Mid = nas
Lowest = nifA
28
Q

Oxygen conundrum (Nitrogen fixation)
/
Protection of nitrogenase against O2 - leghemoglobin

A
  • 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
Q

Rhizobia reduction of ferredoxin

A
  • 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
Q

FixLJ

A
  • 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
Q

Protection of nitrogenase against O2 - heterocyst

A
  • 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
Q

P cluster

A
  • 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
Q

Sources of nitrogen

A
  • 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
Q

NtrB/C

A
  • 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
Q

NtcA

A
  • Analogous to NtrC
  • Controls ammonia repression of nitrate assimilation in cyanobacteria
  • Activated by PipX
  • When ↓ N, NtcA-PipX x PipX-PII
36
Q

AmtB

A
  • 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
Q

Nas genes

A
  • 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

Paper 2 (28 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions