FeS cluster assembly in bacteria Flashcards
Why is iron important?
- Fe is the 4th most abundant element on earth
- high redox potential
important in the following process
1. Cellular respiration
2. Oxygen transport
3. Electron transport
4. DNA synthesis
Name the diffrent most common Ironsulfur Clusters and draw their structures
FeS Cliusters are inorganic compounds which are present in all kingdoms of life
Name the Redox potentials of electrons transport sites in FeS cluster
- FeS clusters are involved in 1e- electron transfer reactions
- Redoxpotential: (-0,43, -0,4, -0,06V)
1. Rubredoxin (einfach) -60mV
2. Ferredoxin (zweifach) -430mV
3. Ferredoxin (vierfach) -400mV
What is the origin of life theory?
1. Origin in an “Iron-Sulfur World”
* Life is believed to have originated in an environment abundant in iron and sulfur, where iron-sulfur clusters were integral to early metabolic reactions and the emergence of life.
2. Favorable Conditions for FeS Cluster Assembly:
* The primordial atmosphere provided suitable conditions for the spontaneous assembly of FeS clusters, which are crucial for electron transfer and catalytic reactions.
3 . Utilization of FeS Clusters in Early Metabolic Reactions:
* Early life forms likely utilized FeS clusters to capture and release electrons in primitive metabolic reactions, enabling essential chemical processes.
4. Evolution of the TCA Cycle from a Reductive Citric Acid Cycle:
* The tricarboxylic acid (TCA) cycle, a central metabolic pathway, might have evolved from an ancient reductive citric acid cycle that utilized two FeS enzymes: aconitase and succinate dehydrogenase.
5. Challenges in an Oxidative Environment:
* As the environment transitioned to an oxidative state, ferrous iron (Fe2+) bioavailability became limited, presenting challenges for early life forms that relied on iron for various metabolic processes.
Why are FeS clusters important?
Fe-S Clusters are essential Cofactors (prosthetic group) and important for e-transfer in
1. cellular respiration
(In Citric Acid Cycle –> Aconitase, Succinat-Dehydrogenase & Electron transport chain –> Protein complex 1,2,3)
2. photosynthesis
3. nitrogen fixation
–> central role of life
name 4 proteins that contain FeS clusters
- Aconitas
- Succinyl-Dehydrogenase
- NADH:Q-Oxidoreduktase (PS 1) and 2,3
- Biotin Synthase
How are FeS Clusters assembled in vitro?
- Synthetic Fe-S clusters self- assemble
Apo forms of the [2Fe-2S] and [4Fe-4S] proteins can be activated by simple addition of HS- and Fe2+/3+ - Why do we need metallochaperone systems for Fe-S cluster assembly?
= To control metabolic resources or to minimize the toxicity of free iron and sulfide
How are metals bound in proteins?
There are 2 main classes of Co-factors
1. Coenzymes (organic molecules derived from vitamins)
2. Metals
Tightly bound coenzymes are called prosthetic group
Enzyme + Cofactore = Holoenzmyme
Enzyme - Cofactor = Apoenzyme
Which Protein is the iron donor in the FeS Cluster biosythesis?
in vivo
- iron (Fe3+) is transported in the cell via siderophores
- it can be released from the siderophore (Fe2+/Fe3+)
- it can be stored in bacterioferritin
- IscA is the Irondonor Protein –> involved in mobilization and transfer of iron for the assembly of FeS Clusters. Transfers Iron to IscU (Scaffolf protein)
Where the actual assembly of FeS occurs
Where does the sulfur comes from?
From the IscS Operon.
- Desulfurase of L-Cys to L-Ala.
- PLP (Pyridoxal-5´-Phosphat) is a cofactor for desulfurases
- PLP is an inactive Form of vitamin B6
- Catalyze the generation of sulfur from L-cystein
Explain briefly th function of PLP (Pyridoxal-5´-Phosphat)
- PLP is kovalently bound to a Lysin residue in the enzyme (Schiff’sche Base, internal aldimin)
- During the reaction this bond is broken and a new bond with the substrate amino acid is formed (external aldimine)
Side Chain cleavage (aldol-cleveage or retro-aldol reaction)
1. Cystein-Desulfurase with coenzyme PLP break down amino acids
2. Amino acids are degraded to compounds that can be metablosed to CO2 and H2O or used in gluconeogenesis and can be the source of the buildign blocks for complicated protein co-factors e.g: cystein desulferase
3. Cystein desulferase - catalyses the conversion of L-Cystein to L-Alanine
4. The Sulfur of Cystein is transferred to many substrates
What Role does PLP has in other Enzymes?
- Decarboxylation of aminoacids
- Transaminations
- Racemizations
- Aldolecleavagereactions
- Transulfurations
Name the FeS cluster biosynthesis operon structure
Scheme: Proposed mechanism for the reaction catalyzed by cystein desulferase
How are the clusters assembled?
- Isc-Operon
* iscR = Regulator
* iscS= Sulfur donor (L-Cystein desulferas)
* icU= Scaffold Protein
* iscA = FeS Cluster insertion
* hscB and hscA= chaperon
* fdx = e-transfer
* iscX = unknwon (maybe Fe-donor CyaY) - There are two steps
- assembly Step
- Transfer step
How are the clusters inserted in the protein?
Cluster transfer:
Fe/S transfer from the scaffold IscU to apo-targets involves HscA and HscB chaperone proteins, which, through ATP-dependent activation, promote a rapid and controlled transfer. For some specific apo-targets, the Fe/S carrier IscA protein is also required for protein maturation.
HscA–HscB System:
* Substrates: ATP and the scaffold protein IscU.
* Recognition: HscA recognizes a specific LPPVK sequence motif on IscU.
* Regulation: HscB is a co-chaperone that interacts with IscU through hydrophobic residues, regulating the interaction between HscA and IscU.
* Role: Facilitates Fe/S cluster transfer from IscU to acceptor proteins.
IscA:
IscA is an Fe-binding dimer protein, contains only three conserved cysteines.
Has a metallic center. Forms [2Fe–2S] or [4Fe–4S] clusters, and binds iron.
- Fe/S-Binding: IscA can form [2Fe–2S] or [4Fe–4S] clusters with complete cysteinyl ligation.
- Fe-Binding: IscA can also bind one iron atom per homodimer, with a strong affinity for iron.
- Function:
1.) Fe/S Carrier: IscA transfe Fe/S-Clusters from IscU and delivers it to apo-target proteins.
2.) Fe-Chaperone: In vivo and in vitro experiments suggest that IscA may act as an iron chaperone for the biogenesis of iron–sulfur clusters under aerobic conditions.
FeS Cluster Release mechanism:
* HscA–HscB Complex: Involves ATP-Fe/S-bound HscA–HscB–IscU complex.
* Mechanisms: Two proposed mechanisms for Fe/S cluster release from free IscU.
How is the synthesis regulated?
IscR: the negative regulator
- transcriptional regulator
- expresses and activates isc and suf transcription, respectively
- contain 3 conserved cystein residues which are requiered for FeS ligation
REMEMBER:
* Promotor + Regulator + Genes = Operon
* repressor regulates Regulator
Negative regulator:
A negative regulator is a molecule, often a protein, that inhibits gene expression. It can work through repressor proteins binding to operator sites,negative feedback loops preventing overproduction, or epigenetic mechanisms like DNA methylation and histone deacetylation. Negative regulation is crucial for controlling the timing and magnitude of gene expression in various cellular processes.
- Gene is on unless repressed (Repressor binds to operator and blocks excess to promoter)
- Gene is off, only activated by repressors that binds to operator and can be deactivated by binding specific Ion to repressor)
What is the role of the “suf-operon?”
SUF: Sulfur Utilization Factor
E.coli makes use of the Suf system when growing under stress conditions, which require the synthesis of stable FeS clusters:
* iron limitation
* oxidative stress
- plays a crucial role, especially when IscR is absent or unable to perform its regulatory function.
- can complement the ISC system in Fe/S cluster biogenesis, ensuring cellular functionality under varying conditions.
SufA: A scaffold protein that facilitates the assembly of Fe/S clusters.
SufB, SufC, SufD: Proteins involved in the formation of the SufBCD complex, which participates in the delivery of sulfur to SufA.
SufS: A cysteine desulfurase that provides sulfur for Fe/S cluster assembly.
How do eukaryotes synthesize FeS Clusters?
In yeast/human:
* ISC-Operon is in the mitochonria
* CIA in the Cytosol and delivers FeS Clusters to nucleus
in higher plants:
* ISC-Operon is in the mitochondira
* CIA machinery in cytosol
* SUF in plastids
The CIA (Cytosolic Iron-sulfur cluster Assembly) machinery consists of 11 Proteins. Its responsible for the assembly of cytosolic and nuclear FeS Cluster, it assembles 4Fe4S clusters on scaffold proteins (often include ISCU1 or ISCU2). This transfer ensures that the FeS clusters reach their intended functional destination. After insertion CIA undergo maturation, adopting ther functional 3d structures. CIA icludes quality controll –> only properly assembled FeS cluster-containing Proteins are functional. FeS (Cofactor) for many proteins, these proteins are involved in diverse processes like DNA repair, ribosome assembly and regulation of gene expression. Chaperons are involved in assembly and release.
Name some FeS cluster containing enzymes and their functions
In Mitochondira:
1. Succinat-Dehydrogenase –> TCA Cycle
2. Complex I, II, III –> electron transport chain
3. Aconitase –> TCA Cycle
In Cytoplasma:
1. Glutamate dehydrogenase
2. Iron regulatory Protein
3. Xanthine dehydrogenase
–> tRNA modification, Translation, Iron regulation
In nucleus:
1. DNA Glycosylase
–> DNA replication, DNA repair, Chromosme segregation, Telomer length regulation
Which Proteins are involved in complex assembly in Eukaryotes?
- ISCU : Scaffolf protein where iron and sulfur are assembled
- NFS1 : provides elemental sulfur
- ISD 11 : Stability of L-Cystein desulfurase
- FXN : Iron availability and regulation of sulfur production
- FDX : provides electron for sulfur reduction (sulfur to sulfide)
What are the main diffrences between eukaryotic and prokaryotic Fe-S cluster biogenesis?
- Presence of ISD11 in eukaryotes (stabilization of cysteine desulfurase NFS1)
- Compartmentalization (eukaryotes: mitochondria, cytoplasm, nucleus
- Regulation and requirement much better understood in prokaryotes (isc, suf, …)
The CIA Pathway
