Shepherd Flashcards
(44 cards)
Ammonia utilisation: Anammox reactions
NH4+ + NO2- –> N2 + 2H2O + energy
N2H4 in anammoxosome which has ladderanes which help to stop diffusion.
4H+ produced for ATP synthesis
The nitrogen cycle
Pool of biologically available nitrogen
NH4 in clay -> nitrate NO3- used by plants
NO3- -> N2 in water logged fields
NO + O3 -> nitric acid/ acid rain
Nitrogen fixation (root nodules)
Rhizobium in root molecules of plants has a mutualistic relationship.
Nitrogen -> ammonia is favourable
Triple N-N bond requires high energy to break
Haber Bosch process
Biological conditions
Equation
Synthetic ammonia was used for the production of HNO2
150-250 bar and 300-550oC
Biological fixation at 0.8bar and biological temp
High activation energy reduced by ATP
N2 + 10H+ + 8e + 16ATP –> 2NH4 + 16ADP + 16Pi +H2
Biological enzymes for ammonia production
Nitrogenase complex structure
Nitrogenase complex- dinitrogenase reductase and dinitrogenease.
DR- dimer of 2 identical subunits, 4Fe-S cluster and ATP binding site on each subunit.
R- 2 types of cofactor. 2 P clusters made up of 4Fe-4S clusters. 2 Fe-molybdenum cofactors (unknown X) also containing S and homocitrate
Nitrogen fixation by nitrogenase complex
Electrons
Ditrogenase reductase- reduces dinitrogenase
Additional 2e used to 2H+ -> H2
8e required per N2 molecule
Binding of 2ATP -300 -> -420
E flow in nitrogenase reaction
Only nitrogenase reductase with 4-Fe4-S in +1 and 2MgATP can associate with nitrogenase (MoFe protein).
2MgATP + e from 4Fe4S –> 2MgADP + e accepted by MoFe.
8 turns of cycle = 2 ammonia
8e = 2 for H2, 6 for 2x NH3
The FeMO cofactor
Site of substrate binding and reduction
Strains deficient are inactive, but reactivated by adding FeMo
In CO inhibited FeMo, shows that CO binds to FeMo cofactor, no to the P cluster
The P cluster (nitrogenase)
Mediate electron transfer between Fe proteins and the substrate reduction site of the FeMo cofactor
p cluster is between 4Fe-4S and the cofactor
AA substitutions between P cluster and FeMo disturb electron transfer.
Nitrogenase complex is oxygen labile
Ditrogenase has a half life of 10 mins
Some bacteria use a respiratory oxidase to burn O2.
Symbiotic supplies nitrogen and bacterial requirements
Engineering of transgenic plants and bacteria to fix N2 (no fertiliser).
Leghaemoglobin
Haem binding protein that scavenges oxygen from bacteroid.
Similar to myoglobin, haeme cofactor.
Functions of NO
Relaxes smooth muscle and acts as a vasodilator.
Activates guanylyl cyclase which makes cGMP
cGMP PK -> myosin light chain phosphatase -> muscle relaxes
NO is toxic to bacteria
Reacts with haeme groups, destroys iron clusters
Can form SNOS and nitrosyl heme
Respiratory inhibition and death
NO + superoxide -> peroxynitrate which can nitrate Tyr residues.
Bacteria encounter NO during infection
Nitrate + stomach acid -> NO
Anaerobic respiration -> NO
iNOS and eNOS
Arg -> hydroxyarginine -> citrulline + NO
What triggers iNOS to release NO?
What are the 3 subunits?
What is the reaction?
Gram -ve covered in LPS, triggers iNOS
1- haeme prosthetic group and BH4
2- FMN and FAD-NADPH as cofactor
3- calmodulin binding group so Ca sensitive
Arginine -> hydroxyarginine->citrulline + NO
Mechanism for NO synthases
cGMP blocks Ca entry to the cell
Ca increases Arg + O2 -> NO
NO then increases cGMP levels
iNOS binds calcium permanently
Bacterial adaptations to NO toxicity
NO -> nitrate by Hmop
NO -> N2O via NorVW
NO is reduced by NrfA
YtfE repairs damaged FeS clusters damaged by NO stress
CydAB allows aerobic respiration under low O2
Hmp detoxifies NO
Equations
HMP-Fe (2)O2+ NO -> HMP-Fe(3) +NO3-
2HMP-Fe (2)NO + 2H+ –> 2 HMP-Fe(3) + N2O + H2O
Catalysed by Fe haeme cofactor
E shuttled from NADH via FAD to reduce iron
Hmp mechanism debate
Debate over O2 first or NO first
May depend on availability of O2 and NO
Flavohaemoglobin regulation
NsrR transcription regulator represses Hmp in low NO
With NO, FeS cluster is nitrosylated and dissociates from DNA
Hmp is transcriptionally regulated by Hcy.
NO -> Inactivates Hcy -> more Hmp expression by metR
NO + 4FeS –> iron species relieve repression.
What is bNOS?
Bacterial NO synthase, gram +ve
Lacks redoxase domain
Probiotic bacteria offer continuous NO supply useful for research
How bNOS increases antibiotic resistance
- chemical modification of toxic compounds by NO
- alleviation of oxidative stress by antibiotics
NO suppresses ROS produced by antibiotics
Inhibition of bNOS research?
Bacteria use hydroxamate to import iron
Siderophores bind iron and transport
Hydroxamate groups strongly bind Fe3+
Iron is then reduced so is useful to cell
Hydroxamate is recycled
What are enterobactins?
Derivatives of catechol
Bind ion with high affinity via 6 O2 atoms
Complex is imported through FepA channel -> peri plasm
FeB chaperone -> cytoplasm via active transport (ATP)
Fes esterase -> free Fe
TonB gate uses PMF (H+ pumped)
