The evolutionary importance of iron sulphur flavoproteins

Iron Sulphur Flavoproteins.

This posting explores how evolution may have brought together flavoproteins and iron-sulphur clusters (as co-factors) to optimise a range of functions – including in sequence. Iron-sulfur clusters rank with biological prosthetic groups, such as hemes and flavins in pervasive occurrence and multiplicity of function.

There has been the tendency in research to explore each of the functions of flavoproteins and iron sulphur clusters separately, but such functions can also combine to support a cycle.

It is proposed that adaptation of multiple functions into a cycle may have emerged through an adaptive evolutionary response – initially to the Great Oxidation Event, and then through endosymbiosis.  Perhaps this even offered the basis for the evolution of photosynthesis and respiration in an oxygen rich environment.

As electron transfer proteins they would enable long range electron transfer (which is used in photosynthesis, respiration, catalytic events). Electron transfer proteins contain redox-active prosthetic groups or “redox sites” where oxidation/reduction occurs. The most common redox sites contain metals such as hemes, iron-sulfur clusters, and copper centers but also include flavins, reducible disulfides, and quinones.  

Electron transfer (ET) is a very simple chemical process but closely governs the life on Earth. In nature, an immediate transfer of the electron(s) in photosynthetic reaction center converts light from the Sun to the forms of chemical energy and is stored in glucose or other types of organic compounds, ultimately supplying “food” for living creatures. In a class of blue-light receptor called cryptochrome found ubiquitously in plants and in animals, the process of ET is pivotal to generate the 24-hour life cycle of circadian rhythm and endogenously informs the body organism “time to sleep”. Furthermore, the ET reaction is also involved in repairing photo-damaged DNA and prevents some diseases like skin cancer. The role of ET in biology serves as an energy transmission and evolves the life using Sun power. Ting-fang He 2011. 

A cycle could included –

  • The generation of reactive oxygen species (ROS)  e.g  Plants and other living organisms in the oxidizing environment constantly produce ROS in chloroplasts, mitochondria, peroxisomes and other sites of the cell because of their metabolic processes such as photosynthesis and respiration.  ROS can also be triggered by various types of environmental stresses.
  • ROS is then modulated through the covalent modification of specific cysteine residues found within redox-sensitive target proteins.  Oxidation of these specific and reactive cysteine residues can then lead to the reversible modification of enzymatic activity.  Reactive and potentially modulatory cysteine residues might exist in many individual proteins, thereby extending this form of redox regulation to a wide range of enzymatic activities.
  • Conformational changes.
  • Signalling Events.
  • Ultra fast electron transfer .
  • A response to oxidative stress ( It has been proposed that these various protein antioxidants might exist in some loose hierarchical network or redox circuit, whose function is to maintain the overall cysteine proteome), redox switching and changes in gene expression..
  • DNA repair (i.e of damage caused by light). 

Perhaps this even offered the basis for the evolution of photosynthesis and respiration in an oxygen rich environment.

Sequential combinations of functions could have also evolved in conjunction with circadian rhythms (including 24 hour redox cycles) which offered a global clock system – ensuring the synchronisation of a sequence of functions within a cycle/system (including the utilisation of various antioxidants). The more complex the cycle, the greater the need for effective synchronisation. The cycle may have developed in different ways within different kingdoms and species (depending on their natural environment, some functions may be emphasised at the expense of another).

Iron-Sulphur Clusters

Iron-sulphur (FeS) clusters are among the most ancient and versatile protein cofactors. They are used by a large and diverse group of proteins, serving both structural and catalytic roles. They act as:

  • Catalytic centres for significant biological processes that require electron transfers e.g photosynthesis, respiration and metabolism and signalling processes governing gene regulation and expression.
  • Electron carriers in redox reactions and have an electron transfer function e.g in photosynthesis, respiration, etc.
  • Sensors of environmental or intracellular conditions (e.g O2, oxidative and nitrosative stress, iron, and metabolic nutritional status) to regulate gene expression accordingly. R Hidese 2014, J Crack 2014, R Lill – ‎2009.
  • by their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. J O Fuss 2015.

Iron-sulphur proteins are found in all life forms. Most frequently, they contain Fe,S, Fe,S4, and Fe,S4 clusters. These modular clusters undergo oxidation-reduction reactions, may be inserted or removed from proteins, can influence protein structure by preferential side chain ligation, and can be interconverted. Their most common oxidation states are paramagnetic and present significant challenges for understanding the magnetic properties of mixed valence systems. H Beinert 1997.S Wollers 2010.

Ferrous iron and sulphur were readily available in the reducing atmosphere in which life first evolved, but when oxygen levels rose with the advent of photosynthetic algae, these building blocks became scarce. Furthermore, the reactive oxygen species generated as byproducts of aerobic respiration are highly damaging to FeS clusters, and free iron and sulfide released by FeS clusters are, in turn, toxic to cells. For these reasons, complex mechanisms evolved to coordinate and regulate the biogenesis of these simple cofactors, and these pathways are compartmentalized in the endosymbiotic organelles of eukaryotic cells… A Teegan 2013, J Crack 2012. Organisms have evolved machinery consisting of specialised proteins that operate together to assemble Fe-S clusters efficiently so as to minimise cellular exposure to their toxic constituents: iron and iron sulfide ions. J H Kim 2015.

Iron Sulphur Proteins

Iron sulphur proteins include the ferrodoxins, rubredoxins, high potential iron proteins and iron-sulphur flavoproteins. Ferrodoxins are found in a wide range of organisms  It has been proposed that there is an evolutionary development of ferrodoxins from the obligate anaerobic bacteria, through the green and red photosynthetic bacteria and the sulphate-reducing bacteria, to the blue-green algae and thence to higher plants and animals.  A F Lodeyro 2012.   This posting explores whether other iron sulphur proteins have undergone an evolutionary development through various species and kingdoms.  This may have seen evolution of early organisms with metabolic pathways that relied direcly on Fe-S clusters through to modern organisms making use of NAD(P)H and NAD (P) and phosphorylation cascades, with Fe-S clusters remaining a common class of enzymatic cofactors.  


Commonalities among flavin based photo and redox receptors could reflect evolutionary relationships among them  E F Yee 2015.

Flavoproteins are ubiquitious proteins in which flavin cofactors play the role of electron transfer intermediate in various biochemical reactions. Many electron transfer reactions in biological systems depend on redox chains that involve flavoproteins.

They display a rich redox chemistry as they can adopt three different redox states (1) oxidised, (2) semi-reduced (radical) and (3) fully reduced. In addition, the redox changes can be accompanied by protonation changes. (A Lukacs 2007). Flavins are arguably one of the most versatile cofactors by virtue of the reactivity of the isoalloxazine ring system. A peculiar behavior of the spin density in some atoms of the flavin ring has been found that could be relevant in understanding reaction mechanisms in flavoproteins. A varied catalogue of reactions for the diverse family of flavoenzymes has been reported, leading to:

  • Flavin cofactors are central to many biochemical transformations and are typically tightly bound as part of a catalytically active flavoenzyme. E A Argueta 2015.
  • Unifying concepts in (long-range) electron transfer, oxygen activation, photochemistry and substrate redox reactions… modes of covalent linkage to flavins, the use of flavins as redox shuttles in microbe mineral electron transfer,have recently been uncovered. D Leys 2016.
  • The properties of flavins provide proteins with a versatile redox sensor that can be utilized for converting physiological signals such as cellular metabolism, light, and redox status into a unique functional output. The control of protein functions by the flavin redox state is important for transcriptional regulation, cell signaling pathways, and environmental adaptation. D F Becker 2011. There are example of flavoproteins which influence gene expression. S Hill 1996,S Metz 2012.
  • Historically photolyases have been described as flavoproteins that repair UV-damaged DNA in a light-dependent fashion.

Flavoproteins are involved in the photosynthetic electron transfer from photosystem I to NADP+.  In addition to their role in photosynthesis, flavodoxin and ferredoxin-NADP(+) reductase are ubiquitous flavoenzymes that deliver NAD(P)H or low midpoint potential one-electron donors to redox-based metabolisms in plastids, mitochondria and bacteria. M Medina 2009. Transfer of electrons between pyridine nucleotides (obligatory two-electron carriers) and hemes or [2Fe-2S] centers (obligatory one-electron carriers) is an essential step mediated by flavins in respiration, photosynthesis, and many oxygenase systems. G T Gassner 1995.  Flavins and iron sulphur clusters also play a role in the respiratory chain, in both Complex 1 E Gnandt 2016  and complex II G Cecchini 2003. A deficiency in the flavoprotein of Arabidopsis mitochondrial complex II results in elevated photosynthesis. D Fuentes 2011

Photolyases and Cryptochromes

The physiological functions of most flavoproteins are light independent, but among the exceptions are a class of proteins comprising of DNA photolyases and cryptochrome (A Lukacs). This posting focuses on this class of flavoprotein and the relationship with iron-sulphur clusters.

Historically photolyases have been described as flavoproteins that repair UV-damaged DNA in a light-dependent fashion, whereas cryptochromes have been described as related proteins without repair activity that serve as photoreceptors or compounds of the inner clock. Cryptochromes are also thought to be involved in magnetoreception. However, more recently, evidence has emerged that has challenged these simple distinctions between photolyases and cryptochromes. e.g Q Mei 2015

The family of photolyases and cryptochromes have been divided into seven major phylogenetic groups: CPD photolyases class I, II and III, Cry-DASH proteins, eukaryotic (6–4) photolyases and animal cryptochromes, plant cryptochromes and prokaryotic FeS-BCP (Fe-S bacterial cryptochromes and photolyases) proteins. D Graf 2015. Animal CRYs are closely related to (6-4) photolyases, while plant CRYs form a sister group of the class III CPD photolyases.  In addition, PHR2 (photolyase/blue receptor 2) subfamily previously reported from green algae Chlamydomonas reinhardtii has been found to be common in higher plants. Results suggest that PHR2 likely evolved from an ancient CRY-DASH gene. Q Mei 2015

Challenges to simple distinctions from bacterial cryptochromes.

Cryptochromes (such as the DASH type or CryA from Aspergillus nidulans), have been found to exert dual functions by being competent in signaling and DNA repair. O Bayram 2008Geisselbrecht – ‎2012. Cyptochrome-DASHs exhibit a variety of physiological functions including single-strand DNA photolyase activity, transcriptional regulation in Synechocystis and light-dependent regulation of metabolism in Fusarium.

Bacterial cryptochromes have been identified in the genomes of some eubacteria including synechocystis sp, cytophaga hutchinsonii and vibrio cholerae. Most knowledge on bacterial cryptochromes has been generated from studies of synechocystis sp PCC6803 cryptochrome and VcCry1 from V Cholerae, belongs to the cry-dash proteins. Investigations of these proteins have not exposed cry functionality towards the organisms, however it appears that they exhibit features of both photolyases (light induced redox activity) and cryptochromes. S Brandt and N Frankenbury-Dinkel edited by Reinhard Krämer, Kirsten Jung 2009.

Bacterial Photolayses – Flavoproteins with Iron Sulphur Clusters.

The group of FeS-BCP proteins (bacterial photolyases) is believed to be the most distantly related to the other members of the cryptochrome / photolyase family. A specific feature of FeS-BCP members is their iron-sulfur (Fe-S) cluster that was thought to be missing in all other photolyases or cryptochromes. This has been more recently challenged.

Examples of FeS-BCP protein members include A. tumefaciens (containing two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster) and CryB in R Sphaeroides (R26) which contains an iron-sulphur cluster as third cofactor (which maybe responsible for its redox-responsiveness). I Oberpichler 2011D Graf 2015Frühwirth 2012.

Results of the mutant studies suggested that PhrB might serve as a photoreceptor for light-regulated motility. The assumption that PhrB could act as photoreceptor is in line with recent finding on the regulation of photosynthesis gene transcription in Rhodobacter sphaeroides by CryB , which is a close homolog of A. tumefaciens PhrB. I Oberpichler 2011

The crystal structures of CryB from Rhodobacter sphaeroides and PhrB from Agrobacterium fabrum (formerly A. tumefaciens C58) have been determined. It has been proposed that the antenna chromophore of PhrB and CryB is 6,7-dimethyl-8-ribityl-lumazine (DMRL), the last intermediate of the flavin biosynthesis pathway before the formation of riboflavin. Other members of the photolyase or Cry-DASH group of proteins have methenyltetrahydrofolate, 8-hydroxy-5-deazariboflavin or flavin mononucleotide as antenna chromophore. D Graf 2015.

Genomic sequence analysis of Acinetobacter Ver3 indicated two genes encoding a class-1 photolyase (PL-1 and another protein (PL-2) which recently showed sequence signatures for a novel subfamily of photolyases that potentially harbor an iron-sulphur cluster as an electron donor. Acinetobacter Ver3, a gammaproteobacterium has the ability to cope with increased UV-induced DNA damage. D Kurthe 2015. The e-coli photolyase (Ec-PL) yielded a nearly perfect match to PL-1 with respect to structure adaptation, and also the location of the cofactors flavin-adenosine-diphosphate (FAD)\and methenyltetrahydrofolate (MTHF) and that of functionally essential amino acids (tryptophan triad).  Albarracin et al. (2012).

mFeS-BCP ( bacterial cryptochromes and photolyases)) sequences have also been found in hundreds of bacterial organisms including many human and plant pathogens such as Vibrio cholerae and Pseudomonas syringae. I Oberpichler 2011P Scheerer 2015.

FeS cluster biogenesis pathways are extremely well conserved, and are invariably essential for viability. Among eukaryotic pathogens, all endosymbiotic organelles studied to date appear to contain FeS cluster biogenesis machinery, and, in some cases, this seems to be the sole reason for retention of the organelle. Typically, the ISC (Iron-Sulfur Cluster formation) pathway resides in the mitochondrion, the CIA (Cytosolic Iron-sulfur protein Assembly) pathway functions in the cytosol and nucleus, and plastid-containing organisms have an additional pathway, the SUF (SUlFur mobilization) system. A Teegan 2013.

There are presently more than 900 prokaryotic PhrB homologs in the database in which the characteristic cysteine residues for incorporation of the Fe-S cluster and other key amino acids are conserved. This new group of (6-4) photolyases with an Fe-S cluster is thus widely distributed among prokaryotes. Unlike in other photolyases, the loop connecting the N-terminal and the C-terminal domains of PhrB interacts with the DNA lesion. A C-terminal extension, which could have a regulatory function, interacts with the DNA. A. T Lamparter 2014.

Common evolutionary origin?

Following the finding that there is a prokaryotic (6-4) photolyase, PhrB from Agrobacterium tumefaciens, it has been proposed that (6-4) photolyases are broadly distributed in prokaryotes, and the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family. F Zhang 2013.


These could potentially have been utilised (through evolution) into a cycle redox sensing – the oxidation of cysteines to cysteine sulfenic acid then reacting with another cysteine residue, to form a disulfide that is subsequently reduced by an appropriate electron donor to complete a catalytic cycle – ultra fast electron transfer – generation of reactive oxygen species (as a messenger)- a response to oxidative stress (including redox switching and changes in gene expression) – and DNA repair (i.e of damage caused by light). 

Supporting the evolution of ultra-fast electron transfer?

Both flavins and iron sulphur clusters have key roles to play in electron transfer. Most biologically relevant photoreceptor proteins contain chromophores that perform photoinduced charge transfer, or an isomerization reaction, as the initial step of a chain of events that leads to a specific type of signalling. Flavin chromophores are an important class of such proteins.

The primary dynamics in photomachinery such as charge separation in photosynthesis and bond isomerization in sensory photoreceptors are typically ultrafast to accelerate functional dynamics and avoid energy dissipation. The same is also true for the DNA repair enzyme, photolyase. However, it is not known how the photoinduced step is optimized in photolyase to attain maximum efficiency. However in the DNA repair enzyme photolyase, it has been found that maximum efficiency was not enhanced by the ultrafast photoinduced process but by the synergistic optimisation of all steps in the complex repair reaction. C Tan 2015.

Very fast photo-induced electron transfer is feasible along the conserved tryptophan pathway found in flavoproteins of the photolyase/cryptochrome family.  Both cryptochromes and photolayses are flavoproteins that undergo ultrafast charge seperattion upon electron excitation of theif Flavin cofactors.  T Firmino 2016. Dr T Biskup 2011.  Ya-Ting Kao 2008,  D Zhong 2006.

A relationship between ultra fast electron transfer and quantum coherence

Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity.M Ferretti 2016  It has been suggested that during photosynthesis, plants use electronic coherence for ultrafast energy and electron transfer and have selected vibrations to sustain those coherences.  In this way photosynthetic energy transfer and charge separation have achieve their amazing efficiency.  At the same time these interactions are used to photoprotect the system against unwanted byproducts of light harvesting and charge separation at high light intensities. Rienk van Grondelle 

Possible evolutionary relationship with Complex I and II?

Complex I contains one non-covalently bound flavin mononucleotide and, depending on the species, up to ten iron-sulfur (Fe/S) clusters as cofactors. The reason for the presence of the multitude of Fe/S clusters in complex I remained enigmatic for a long time. The question was partly answered by investigations on the evolution of the complex revealing the stepwise construction of the electron transfer domain from several modules. Extension of the ancestral to the modern electron input domain was associated with the acquisition of several Fe/S-proteins. The X-ray structure of the complex showed that the NADH oxidation-site is connected with the quinone-reduction site by a chain of seven Fe/S-clusters. Fast enzyme kinetics revealed that this chain of Fe/S-clusters is used to regulate electron-tunneling rates within the complex. E Gnandt 2016.

One feature of the complex II structures is a linear electron transport chain that extends from the flavin and iron-sulfur redox cofactors in the membrane extrinsic domain to the quinone and b heme cofactors in the membrane domain. G Cecchini 2003

Bioinformatics have been used to investigate the human portfolio of FeS-Ps (iron sulphur proteins)…comparative analysis of the organisation of the prokaytoic homologues of human FeS-Ps within operons allowed reconstruction of the human functional networks involving conserved FeS-Ps commons to prokaryotes and eukaryotes. These functional networks have been maintained during evoluation and thus presumably represent fundamental cellular processes. The respiratory chain and the ISC machinery for FeS-P biogenesis are the two conserved processes that involve the majority of human FeS-Ps….The analysis of the co-occurence of human FeS-{s with other proteins highlighted numerous links between the iron-sulphur cluster machinery and the response mechanisms to cell damage, from repair to apoptosis. This relationship probably relates to the production of reaction oxygen species within the biogenesis and degradation of FeS-Ps. C Andreini 2016

Cysteine Residues and Ultra-Fast Electron Transfer

In  order to understand reactive oxygen species ROS) regulation of signaling pathways it is necessary to understand the mechanism of how ROS alters protein function. The oxidative interface consists mainly of the redox regulation of redox-reactive cysteine residues on proteins by ROS. Findings indicate that the cysteine residues exposed on the surface of proteins are the dominant intracellular thiol and that they may play an important role in intracellular antioxidant defences. R Requejo 2010.  By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis.  Oxidative modifications result in changes in structure and/or function of the protein.  P D Ray 2012.

It has been proposed that the side chains of tyrosine and cysteine residues can act as the relay stones of electron transfer in proteins.  The driving forces come from the loss of the active protons (protons link the ring oxygen of the tyrosine and the sulphur of the cysteine) in the appropriate protein environments, which lowers the reduction potentials of these two residues.  An example is the long distance electron transfer of the class I ribonucleotide reductase, involving four tyrosines and a cysteine residue. Additionally, the Giese Group, examined electron transfer along a series of polypeptides and demonstrated that the existence of central aromatic acids can serve as stepping stones to support the electron hopping mechanism. W Sun 2016.  

And it has been reported that the side chains of tryptophan residue can speed up electron transfer rates as relay intermediates of long range electron transfer processes in many enzymes including DNA photolyases, where three Trp residues can form an electron transfer wire to facilitate electron hopping. W Sun 2016.  In the complex 1 of the respiratory chain on the inner membrane of the mitrochrondria, electrons are transferred over a cascade of iron-sulphur clusters from a Flavin adenine mononucleotide cofactorto a quinone binding set.  The overall pathway is nearly 10nm long and most likely to involve aromatic amino-acid residues as stepping stones to bridge the gaps between the iron-sulphur clusters.

It is interesting to note that extended electron-transfer in animal cryptochromes in mediated by a tetrad of aromatic amino acids.  D Nohr 2016.  Some photoreactive proteins, including those responsive to UV light, utilize characteristic aligned tryptophan residues for electron transfer.  Very fast photo electron transfer (PET) is feasible along the conserved linear tryptophan pathway found in the flavoproteins of the photolyase/cryptochrome family, including Arabidopsis UVR2 and UVR3…starting from the singly reduced (semiquinod) state of FAD, the overall photoreduction process is completed within 30ps, as has been shown by time-resolved optical absorption spectroscopy in E Coli DNA photolyase.  Dr T Biskup 2011.

For FAD in the fully oxidized state, PET generates a sequence of radical pair species. And in flavo-proteins (mutated in order to remove a cysteine residue next to the chromophore), it is possible to form a spin-correlated radical pair. Such radical pairs recombine, generally speaking, from both singlet and triplet states, giving rise to strong solid state-CIDNP. Because of the rigid environment, molecular mobility is strongly restricted; therefore, anisotropic spin interactions come into play and affect the evolution of the radical pairs… Presently, flavoproteins represent the only system, where both liquid state-CIDNP and solid state-CIDNP have been observed (even then, for slightly different flavoproteins).  Denis V. Sosnovsky.  2016. 

Commonalities among flavin-based photo and redox receptors could reflect evolutionary relationships among them.  In the canonical LOV photocycle, a Flavin excited triplet state reacts with the thiol group of a conserved cysteine residue.  Bond formation likely proceeds via a redox process as supported by detection of a transient Flavin neutral semiquinone (NSQ) in C reinhardti phot1 LOV1, by indirect arguments from magnetic resonance experiments and by the general efficacy of Flavin photoreduction in the cysteine-devoid variants. Thus the NSQ is a likely intermediate in generating the adduct.  The BAT-LOV P1988C variant demonstrates that cysteines at the adduct forming position is an effective electron donor to the photo excited Flavin.  LOV signalling through the NSQ state has parallels to signal transduction in other flavin based photoreceptors including cryptochromes and BLUF.  There is strong evidence that the signalling state of cryptochrome involves reduction of the FAD to either NSQ or anionic semiquinone states.  A NSQ state may be populated transiently during the photocycle of a radical pair intermediate between the Flavin and a conserved Try. . E F Yee 2015.  Also see A Hense 2014.   A Czarna2013. R J Kutta 2015 for the role of cysteine residues in the generation of radical pairs in various flavoproteins.

Orf et al 2016 has recently demonstrated that an operative photoprotection mechanism exists in green sulfur bacteria and that this mechanism is activated by oxidation of two cysteine residues.  This new photoprotection mechanism identified by differs from more familiar motifs; the new mechanism employs amino acid residues instead of isomerization of dedicated photoprotective chromophores, such as carotenoids. It also seems to protect against damage from a single excitation (rather than multiple excitations). That is, the mechanism depends on redox potential, not light intensity….The system undergoes intersystem crossing into the triplet state after ~25% of excitations, and the lifetime of the triplet state is between 10-100 μs. The redox-sensitive cysteine residues in the protein,  modulate their redox state between free thiol and thiyl radical form to quench BChl excitations, probably via an electron transfer/ultrafast recombination mechanismOrf 2015.

Regulation of Gene Expression

As the following table on ‘Fe–S proteins that regulate gene expression’, taken from E L Mettert 2015 demonstrates, iron sulphur proteins can influence gene expression – as an adaptation to oxidative stress.

Table 1.
        Fe–S proteins in the regulation of gene expression.
Regulator Organism Cluster type Primary signal Function
FNR Proteobacteria and Bacilli [4Fe–4S] O2 Controls genes in the adaptive response to anaerobiosis
NreB Staphylococci [4Fe–4S] O2 Phosphorylates the response regulator NreC to control genes in nitrate/nitrite respiration
AirS Staphylococcus aureus strain Newman [2Fe–2S] O2 Alters anaerobic expression of genes involved in quorum sensing, virulence, comma, and oxidative stress
SoxR Proteobacteria and Actinobacteria [2Fe–2S] Redox-cycling compounds Regulates the oxidative stress response directly or through the transcription factor SoxS
IscR Proteobacteria [2Fe–2S] Fe–S cluster levels Controls genes in Fe–S cluster biogenesis
SufR Cyanobacteria [4Fe–4S] Fe–S cluster levels Controls the sufBCDS Fe–S cluster biogenesis pathway
RirA Rhizobia Not yet determined Fe levels Regulates genes in Fe uptake
Fra2–Grx3/4 Saccharomyces cerevisiae [2Fe–2S] Fe levels Controls activity of Fe-uptake regulators Aft1/2
Aft1/2 Saccharomyces cerevisiae [2Fe–2S] Fe levels Regulates genes in Fe uptake
IRP1 Mammals [4Fe–4S] Fe levels Alters stability or translation of transcripts involved in Fe uptake
Bacterial aconitases Bacteria [4Fe–4S] Fe levels and/or ROS/NO Alters stability or translation of transcripts, including those involved in Fe homeostasis, the oxidative stress,  response, motility, or sporulation
NsrR Proteobacteria<comma> Bacilli<comma> Streptomyces [2Fe–2S] or [4Fe–4S] NO Regulates genes in the NO stress response
Wbl proteins Actinobacteria [4Fe–4S] NO Regulate genes involved in diverse cellular processes, including development, virulence, or antibiotic resistance
ArnR Corynebacterium glutamicum Not yet determined NO Controls genes in nitrate metabolism
RsmA Streptomyces coelicolor [2Fe–2S] Not known Inhibits activity of σM
ThnY Sphingomonas macrogolitabida [2Fe–2S] Not known Promotes activity of the tetralin utilization regulator ThnR
VnfA Azotobacter vinelandii [3Fe–4S] Not known Transcriptional activator of nitrogenase-2

The appearance of oxygen in the atmosphere led to the development of defence mechanisms that either kept the concentration of the O2-derived radicals at acceptable levels or repaired oxidative damages. Iron plays a significant role in biology (transport, storage and activation of molecular oxygen, reduction of ribonucleotides, activation and decomposition of peroxides, and electron transport) and Fe2+ is required for the growth of almost all living cells. Yet potentially toxic iron-catalyzed reactive oxygen species (ROS) are unavoidable in an oxygen-rich environment. Iron and ROS are increasingly recognized as important initiators and mediators of cell death in a variety of organisms and pathological situations. S J Dixon 2013. In addition highly reactive species can be generated from flavoproteins, ROS formation results from cry1 activation and induces cell death in insect cell cultures. In plant protoplasts, cryptochrome activation results in rapid increase in ROS formation and cell death. L Consentino 2015. Direct transfer of a single electron by oxygen by reduced enyzmes or prosthetic groups, such as flavoproteins and iron sulhpur clusters is a source of superoxide (a reactive oxygen species).

It is asked in this posting whether flavins and iron sulphur clusters could have been brought together (as co-factors) to negate their generation of damaging reactive oxygen species, and instead act to reduce oxidative stress. There area number of examples where combinations of flavoproteins with iron sulphur clusters have been shown to be participating in oxidative stress management.

To reduce the deleterious effects of reactive oxygen species, most bacteria have evolved specific sensor proteins that regulate the expression of enzymes that detoxify these species and repair proteins. Some bacterial transcriptional regulators containing an iron-sulfur cluster are involved in coordinating these physiological responses. Mechanistic and structural information can show how these regulators function, in particular, how chemical interactions at the cluster drive subsequent regulatory responses. The [2Fe-2S] transcription factor SoxR (superoxide response) functions as a bacterial sensor of oxidative stress and nitric oxide (NO). K Koboyashi 2014. Results suggest that any concentration of oxygen free radicals inside cells is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters. G P Riboldi 2014.

However any oxidative stress system must also be able to strike a balance when responding to reactive oxygen species, which also plays a role in a variety of cellular process, including conferment of tolerance to environmental stresses. Reactive oxygen species can also act as a important biological messenger.

The iron sulphur flavoprotein could deliver this through initially acting as a redox sensor (detecting the presence of redox active molecules in the environment, and those produced by metabolic process in the cell). Redox sensors will then control the expression of approriate adaptive responses. Such sensors coould held coordinate the switch between anaerobic and aerobic lifestyles. Reactive oxygen and nitrogen species, produced endogenously or present in the environment, are also important signal molecules that can be readily sensed by their redox activity. This suite of sensor proteins is as diverse as their activating signals. Some sensors contain redox active metal centers that may include mononuclear iron, iron-sulfur clusters, and heme cofactors. Other redox switches rely on the facile oxidation and reduction of bound flavin cofactors or cysteine thiolates to effect changes in protein activity. J D. Helmann 2013

An iron sulphur flavoprotein from Methanosarcina thermophila, which participates in oxidative stress management by removing oxygen and hydrogen peroxide… The protein was eventually found to catalyze the reduction of dioxygen and hydrogen peroxide to water, and its role in combating oxidative stress in strictly anaerobic prokaryotes had been proposed. Many anaerobic bacteria possess multiple Isf paralogs, and it was suggested that these proteins perform for anaerobes universally important and possibly diverse function… Also given the presence and properties of the redox cofactors, it has been suggested that Isf play a role in electron transport, possibly functioning as a one- to two-electron switch… The only eukaryotic species found to possess an Isf homolog in its genome was the anaerobic intestinal pathogen Entamoeba histolytica . The bacterial-type Isf protein present in T. vaginali shydrogenosomes could reduce a variety of substrates, all of them being molecules toxic for anaerobic microbes.  T Smutná – ‎2014.    

The Isf2 gene in A Fulgidus is clustered with genes encoding oxidative stress proteins. S L A Andrade 2005.

CryB in Rhodobacter sphaeroides has been shown to undergo a photocycle it does not only affect gene expression in response to blue light illumination but also in response to singlet oxygen stress condition. Geisselbrecht – ‎2012Hendrischk et 2009. The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA (which has dual sensing capability – integrating both redox and light signals) and CryB, have been identified in the past, which are involved in this regulation. S Metz 2012. Also see C Bauer 2003.  Rhythmic gene expression is common to many kingdoms and species.

Iron limitation increases ROS levels in R Sphaeroides, and it has been found that the link between iron availability and oxidative stress exists for only a subset of iron-dependently regulated genes. The OxyR regulator provides a link between the responses to oxidative stress and iron.  IscR of Rhodobacter sphaeroides functions as repressor of genes for iron-sulfur metabolism and represents a new type of iron-sulfur-binding protein. B Remes 2014.

The above strategies forms part of a facultatively phototrophic bacteria (like rhodobacter) ability to adapt quickly to changing environmental conditions, using different pathways for energy conversion. If high levels of oxygen are present, rhodobacter gains energy by aerobic respiration and synthesizes only low amounts of photosynthetic complexes, however if the oxygen tension drops below a threshold value, the synthesis of pigments and pignment binding proteins is strongly increased and photosynthetic complexes are assembled into a newly formed intracytoplasmic membrane system leading to an increated in pigmentation in the cultures. At intermediate oxygen levels formation of photosynthetic complexes is represesed by light in order to avoid the formation of singlet oxygen. G Klug. 407 singlet oxygen responsive genes have been found (Glaeser 2007, and B A Berghoff and G Klug 2016).

It is asked whether the above findings on iron sulphur clusters, as has implications for response to magnetic fields.

In the case of the photosynthetic bacteria Rhodobacter sph, under conditions that enforce electron back-transfer, the reaction processes are also affected by external magnetic fields. These magnetic field effects originate from the hyperfine coupling in the radicals but, more interestingly, depend also on other spin-dependent interactions characteristic of the primary electron transfer in photosynthesis….the yield of triplet products generated in the reaction centers of Rhodobacter sph. is lowered by external magnetic fields. K Schulten 1977. A magnetic field of 20 millitesla, just 400 times the Earth’s magnetic field, is enough to cut singlet oxygen production by up to 50%. Under this magnetic field, the photosynthetic molecules were protected against singlet oxygen damage. Liu Y et al 2005.  Also see P Braun 2005. The radical-pair mechanism, by which a magnetic field alters the product yields of radical-pair reactions, is by now well established theoretically and experimentally (Salikhov et al., 1984, Steiner and Ulrich, 1989). Magnetic field effects have been studied on radical-pair systems in solution (Schulten et al, 1976, Werner et al., 1977, Haberkorn, 1977, Schulten and Weller, 1978) and in a bacterial photosynthetic reaction center (Werner et al., 1978, Haberkorn and Michel-Beyerle, 1979; see also reviews (Hoff, 1981, Schulten, 1982, Boxer et al., 1983]. Changes in product yields by magnetic fields of 10–100 G are well-documented as reviewed in Steiner and Ulrich (1989)). T Ritz 2000.

Similar effects (categorised as solid state photo-CIDNP) are seen in:

  • Green Sulphur Bacteria (Roy et al 2007). Also see S Kihara ‎2015.  Orf et al 2016Orf et al 20162015  has recently demonstrated that an operative photoprotection mechanism exists in green sulfur bacteria and that this mechanism is activated by oxidation of two cysteine residues. The new mechanism employs amino acid residues instead of isomerization of dedicated photoprotective chromophores, such as carotenoids. It also seems to protect against damage from a single excitation (rather than multiple excitations). That is, the mechanism depends on redox potential, not light intensity….The system undergoes intersystem crossing into the triplet state after ~25% of excitations, and the lifetime of the triplet state is between 10-100 μs. The redox-sensitive cysteine residues in the protein,  modulate their redox state between free thiol and thiyl radical form to quench BChl excitations, probably via an electron transfer/ultrafast recombination mechanism.
  • Spinach (Alia et al 2004, Diller at al 2007, Matysik et al 2000, Diller et al 2007, Diller at al 2005).
  • the photochemical yield of a flavin-tryptophan radical pair in Escherichia coli photolyaseK B Henbest 2008,
  • A mutant of the bluelight photoreceptor phototropin (LOV1-C57S) from Chlamydomonas reinhardtii). S S Thamarath 2010.  In flavo-proteins (mutated in order to remove a cysteine residue next to the chromophore), it is possible to form a spin-correlated radical pair. Such radical pairs recombine, generally speaking, from both singlet and triplet states, giving rise to strong solid state-CIDNP. Because of the rigid environment, molecular mobility is strongly restricted; therefore, anisotropic spin interactions come into play and affect the evolution of the radical pairs… Presently, flavoproteins represent the only system, where both liquid state-CIDNP and solid state-CIDNP have been observed (even then, for slightly different flavoproteins).  Denis V. Sosnovsky. 2016.

In the same way that photo-CIDNP MAS NMR has provided detailed insights into photosynthetic electron transport in Reaction Centres, it is anticipated in a variety of applications in mechanistic studies of other photoactive proteins. It may be possible to characterize the photoinduced electron transfer process in cryptochrome in detail. W Xiao-Jie12016.

It has been noted there seems to be a link between the conditions of occurrence of photo-CIDNP in RCs and the conditions of the unsurpassed efficient light-induced electron transfer in RCs. J Matysik 2009, I F Cespedes-Camacho and J Matysik 2014

Such an effect caused through the interaction of redox and circadian rhythms (potentially involving cryptochrome/photolyases and iron sulphur clusters) may offer a form of antioxidant – reducing singlet oxygen production.

Reaction centres from plants’ photosystem II share many features with bacterial reaction centres, including a high-spin iron whose function has remained obscure. Based on experimental observations that the high-spin Fe2+ ion affects photosynthetic radical pair reactions, it has been proposed that spin plays a direct role in contributing towards the prevention of destructive events in R Sphaeroides photosynthetic reaction centres (and possibly plant reaction centres). This raises the question of whether the effective magnetic field generated by a fast-thermalising spin may play a role in other biological processes, particularly those where magnetic field effects have been observed. Marais et al 2015.

The above strategy might also support both photosynthesis and magnetoreception (I K Kominis 2013A.T. Dellis, I.K. Kominis 2011Robert J. Usselman 2014, and Dr. Matthew Goodman 2010Eugenio Daviso 2009) – and so be integrated into (and perhaps emerging from) that wider circadian-redox-antioxidants cycle (R Hardeland and T Vanden Driessche 2000).

Other redox/circadian interactions (e.g through peroxiredoxin) might also be implicated in solid state photo-CIDNP and ultra fast electron transfer.  Antioxidant peroxiredoxin proteins, which have probably evolved from a thioredoxin‐like ancestor, are present in almost all living organisms, and have been identified as conserved markers for 24‐hour rhythms, also target of cysteine residues. In addition to cyclic oxidation of peroxiredoxin proteins, it is highly likely that redox oscillations impact directly on many other susceptible proteins in cells. Specifically, so‐called hyper‐reactive cysteine residues represent particularly attractive targets. Peroxiredoxin proteins may not be unique in their ability to undergo redox oscillations since may other proteins are susceptible to oxidation of their cysteine residues by peroxide.  S Ray 2016.  

Peroxides oxidize the peroxidatic cysteine  to cysteine sulfenic acid , which then reacts with another cysteine residue, named the “resolving” Cys (CR) to form a disulfide that is subsequently reduced by an appropriate electron donor to complete a catalytic cycle. 

Supporting the evolution of a magnetoreceptor?

In November 2015, scientists announced that they discovered a polymer-like protein, dubbed MagR (Drosophila CG8198), and determined that it forms a complex with a photosensitive protein called Cry. The MagR/Cry protein complex, the researchers found, has a permanent magnetic moment, which means that it spontaneously aligns in the direction of external magnetic fields. This is the only known protein complex that has a permanent magnetic moment. Cry likely regulates the magnetic moment of the rod-shaped complex, while the iron-sulfur clusters in the MagR protein are probably what give rise to the permanent magnetic polarity of the structure. S Qin – ‎2016.

Iron Sulphur Cluster Proteins and DNA repair

Widespread in nature, Fe–S proteins participate in gene regulation. but have prominent functions in several diverse biological processes, including respiration, photosynthesis, nitrogen fixation, RNA modification, and DNA replication and repair. In the case of bacteria, many regulatory proteins coordinate gene expression in response to specific environmental cues, which are often sensed through their co-factors, to enable rapid adaption. Bacterial iron–sulfur cluster proteins can function as regulators of gene transcription. Because clusters of different nuclearity and shape are able to interconvert, they can act as powerful drivers of protein conformational change. In all cases, the cluster acts as a sensor of the environment and enables the organism to adapt to the prevailing conditions, leading directly to changes in DNA binding affinity. 

Iron Sulphur clusters might also be implicated in epigenetic changes mediated through host-parasite relationships e.g NsrR is a global regulator of the response to NO-induced stress and has been shown to be important for establishing symbiosis between Vibrio fischeri and its squid host. NsrR belongs to the largely understudied Rrf2 family that includes the [2Fe-2S] cluster-containing regulator IscR. J C. Crack 2012. E L Mettert 2015.

An evolutionary relationship between prokaryotic photolyases and PriL?

It has been proposed that the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family. Y Geisselbrecht 2012F Zhang 2013,D Graf 2015,

Phylogenetic studies place PhrB next to PriL, indicating an early branchpoint of FeS-BCP and other cryptochrome / photolyase family members. Dominik Graf 2015

PriL (the large subunit of eukaryotic and archaeal primases), and photolyases share a common protein fold. PriL also contains an Fe-S cluster, which is located within the common fold at the same position as the Fe-S cluster of FeS-BCP proteins. A conserved Fe-S domain is necessary to initiate DNA replication.C Zhang 2014. A structural homology between the catalytic domain of Arabidopsis thaliana cryptochrome 3 and the Fe/S-cluster subdomain of the archaeal/eukaryotic primase subunit PriL has been shown, suggesting a plausible evolutionary relationship. Y Geisselbrecht 2012.

Structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/ cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase. (Sauguet et al 2010).

PriL-CTD contains a 4Fe-4S cofactor. The iron-sulphur centre seems to have a structural role in maintaining the C-terminal sequence of PriL in its correct three dimensional shape. The functional role of the Fe-S cofactor depending on its redox status remains speculative, but can not be ruled out. The molecular mechanism underlying PriL-CTD’s involvement in primer synthesis is unclear, but an intriguing clue as to how this might happen comes from the unexpected similarity between PriL-CTD and the active site of DNA photolyase/cryptochrome family of DNA repair enzymes.

The mode of binding of single stranded (ss) DNA and flavin adenine dinucleotide (FAD) observed in the co-crystal structure of the DASH cryptochrome 3 from Arabidposis Thaliana (Pokorny et al 2008) suggests that the PriL-CTD could adopt a similar mode of interact with the template DNA and ribonucleotides during de novo RNA synthesis. In particular, the special relationship between FAD and the extruded, cross linked pyrimidine dimer observed in the active site of DNA photolayse suggests a possible arrangement for the pairing of the first dinucleotide of RNA primer onto template DNA during the initiation step catalyzed by the primase. Thus, the PriL-CTD might participate in the RNA primer synthesis by assisting the catalytic subunit PriS in the simultaneous binding of the two initial RNA nucleotides and by promoting dinucleotide base-pairing with template DNA at the initiation site.  L Pellegrini (in the Eukaryotic Replisome – edited by S MacNeil 2012).

A surprisingly high number of proteins involved in DNA replication and repair have been identified to bind to an iron-sulphur (FeS) cluster. Maturation of FeS proteins is a multi-step process that takes place in mitochondria and the cytoplasm. How it is linked to nuclear proteins has remained unclear, Gari 2012.

Combining circadian rhythms and DNA repair

It has also been suggested that DNA repair might have a common evolutionary origin with circadian rhythmicity. G W, Rosbash (2003). Circadian rhythmicity and photo-activated DNA repair were suggested to have a common evolutionary origin.  Sancar A (2000). Escape from sunlight represented a major selective force for development of circadian rhythms. Q Mei 2015.

Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses. Mammals possess no CPD class II photolyases, except for marsupials (Monodelphis domestica and Sarcophilus harrisii). Recent research demonstrated that the CPD photolyase in a marsupial Potorous tridactylus was able to act as a cryptochrome, suggesting that ancestral CRY/PHR proteins were likely manifested both DNA repair and circadian clock function. I Chaves 2011.  M A Biernet 2012.

The zebrafish circadian clock is controlled by a cell-autonomous and light-dependent oscillator, where zCRY1a functions as an important mediator of light entrainment of the circadian clock. Light directly activates MAPK signaling cascades in zebrafish cells and there is evidence that light-induced activation of these pathways controls the expression of two evolutionary-related genes, z64Phr and zCry1a, revealing that light-dependent DNA repair and the entrainment of circadian clock share common regulatory pathways. J Hirayama 2009

There had been no evidence for common ancestor of eukaryotic and prokaryotic circadian genes, until it was found that the cryptochromes have a common ancestor with the prokaryotic photolyase. M Ahmad 1993. However, it is still unclear what circadian function is performed by CRY-DASH in prokaryotes. Another open question is how the distinctive circadian mechanisms emerged in different groups of eukaryotes. Q Mei 2015.

It has been proposed that early metazoans avoided irradiation by descending in the oceans during the daytime. Bue-light photoreception evolved in an aquatic environment(perhaps because only blue light can penetrate to substantial depths in water). These photoreceptors were then also critical for sensing the decreased luminescence that signals the coming of night and the time to return to the surface. The oceans and the 24-h light-dark cycle therefore provided an optimal setting for an early evolutionary relationship between blue-light photoreception and circadian rhythmicity. W Gehring 2003.

The coupling of cryptochrome and iron sulphur clusters could also potentially be supported by an emerging link found between circadian rhythms and the metabolism/redox. . Lisa Wulund 2015, A Stangherlin – ‎2013, K Nishio 2015, N B Milev 2015, A Ribas-Latre 2016.  There is also a growing realisation in biology that transcription based clocks do not operate in isolation, but rather are mutually dependent upon intrinsically rhythmic cytosolic signals (cAMP, Ca2+, kinases) such as that the cell as a whole has a resonant structure tuned to 24 hour operations (Hastings et al 2008 in C Colwell 2015).

The foundations of the coupling between redox and the circadian system may have been laid down around the time of the Great Oxidation Event (GEO) approximately 2.5 billion years ago. The increase in atmospheric oxygen levels as a result of the newly acquired ability of photosynthetic bacteria to use water as the main electron donor are thought to have created a strong selective pressure on anaerobes to evolve defense systems to deal with this harsh and unprecedented oxidizing environment. Rhythmic photosynthesis, and thus oxygen production as a function of the changing day and night, as well as the generation of reactive oxygen species (ROS) by metabolic reactions, or directly by UV radiation, could have forced the coevolution of the circadian and redox systems. Thus, the generation of ROS and those processes sensitive to oxidation were temporally segregated, preventing harmful oxidative stress that would otherwise have led to cell dysfunction and death….Temporal separation of cellular metabolism might be an adaptation to prevent the simultaneous occurrence of mutually-antagonistic reactions that would otherwise result in energetically-wasteful futile cycles.  N B Milev 2015.

It has also been suggested that circadian clocks may share a common ancestry. Since the cyclical changes occur in a metabolic protein that cleans up reactive oxygen species, it has been propose that sensing and responding to the accumulation of oxygen in the atmosphere 2.5 billion years ago could have driven the evolution of circadian rhythms. A family of antioxidants called peroxiredoxin proteins, which mop up excess hydrogen peroxide in the cell in 24-hour oxidation-reduction cycles linked to metabolism. The proteins, which are found in virtually every organism, have been shown to exhibit circadian oscillations in human, mice, and marine algae cells. R S Edgar 2012. In view of the proposed evolution and functions of photolyase and cryptochrome, it would seem surprising if there was not some link between them and peroxiredoxin.

Photolyases have already played a major role in the development of our understanding of DNA repair mechanisms. In addition:

  • The circadian clock and the DNA damage response are two global regulatory mechanisms that control many aspects of cellular physiology and adaptation to the environment at the cellular and organismal level. The two regulatory systems can operate independently of one another. However, under physiological conditions, the two systems interface. As a consequence, a signal that primarily affects one system, to varying degrees, ultimately affects the other as well…It is conceivable that the last common ancestor of the present day photolyase/cryptochrome family was the blue light sensor directing the diel vertical movement as well as using the blue light as an energy source to repair the UV-induced DNA damage that occurred under those conditions. This primitive flavoprotein with blue-light absorption maxima then diverged to give rise to the present day photolyases which repair DNA and to cryptochromes that control the circadian clock by light-dependent and light-independent mechanisms.
  • Even though photolyase in mammals no longer has a photosensory function and cryptochrome no longer has a direct DNA repair function, cryptochrome still participates in the maintenance of genomic integrity against DNA damage induced by UV and UV-mimetic agents. Cryptochrome contributes to DNA repair/genome maintenance by regulating nucleotide excision repair which is the sole repair mechanism for repairing UV-induced photodimers in placental mammals which lack photolyase, and by coordinating the circadian clock with DNA damage checkpoints which also aids in cell survival and hence in “escape from light. A Sancar 2010. While they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. Results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation. S J Papp 2015.
  • In addition to transcriptional drive, the positive arms of the TTFL are now known to have DNA remodelling activity. DNA is normally packaged into dense chromosomes by special proteins called histones, which can be modified chemically to compact and decompress DNA so that it is inactive or active respectively. CLOCK, for example, is known to function as a histone acetyltransferase (Doi et al., 2006), and this acetylation is required for the rhythmic expression of other core clock and output genes, including its partner BMAL, which undergoes rhythmic acetylation in the liver (Hirayama et al., 2007a). L Wulund 2015.
  • Many organisms possess both a cell cycle to control DNA replication and a circadian clock to anticipate changes between day and night. In some cases, these two rhythmic systems are known to be coupled by specific, cross-regulatory interactions. Mathematical modeling suggests the regular, discrete jumps in gene-copy number arising from DNA replication during the cell cycle cause a periodic driving of the circadian clock, which can dramatically alter its behavior and impair its function. But biological organisms are likely to have developed additional strategies to mitigate this effect. J Paijmans 2015. 

Flavoproteins with iron sulphur clusters might have influenced the emerging linkage being made between circadian rhythms and metabolism/redox?

For example could they have influenced the evolution of:

  • Light dependent protein A (LdpA) a component of the cyanobacterial circadian clock, has been proposed to act as a redox sensor and to be used by the clock to adjust the period length. LdpA contains iron-sulfur centers and can sense the redox state of the cell, which correlates with the amount of light (high light correlates with a reduced redox state, whereas low light is associated with an oxidized redox state. It is also interesting to note that phylogenetic analysis showed that sll1629 (of cyanobacterium synechocystis) is more closely related to the cryptochromes than photolyases, and it has been concluded that sll1629 is a bacterial cryptochrome. K Hitomi 2000. Lack of Syn-CRY cryptochrome in Synechocystis 6803 (sll1629) retards PSII repair. Syn-CRY is required for efficient restoration of Photosystem II activity following UV-B and PAR induced photodamage. This effect is not caused by retardation of DNA repair, instead the synthesis of new D1 (and D2) subunit(s) and/or the assembly of the Photosystem II reaction center complex is likely affected due to the lack of intracellular CO2, or via a so far unidentified pathway that possibly includes the PilA1 protein. I Vass 2014.
  • The effects of altered ROS and the circadian clock have also been observed in N. crassa and in the cyanobacterium Microcystis aeruginosa, in which HO has been shown to impact on the daily expression pattern of clock genes as well as clock-controlled genes, including those involved in coordinating photosynthesis. These results clearly show that fluctuations in the redox state of the cells have an impact on the expression of clock-related genes in multiple diverse systems.   A Stangherlin – ‎2013.    M Katayama – ‎2003.   NB Ivleva 2005, NB Milev – ‎2015 .
  • The Cryptochrome protein (CRY) in Arabidopsis, Drosophila, and mouse provide the most direct path by which redox status can interact with the core components of the transcription–translation feedback loop (TTFL). CRY shares homology with a phylogenetically ancient enzyme family that repairs DNA in response to ultraviolet light known as the photolyases, leading to speculation that redox is therefore crucial to CRY function. Indeed, the CRY protein does contain motifs that bind the flavins, a group of organic compounds known for their prominent role in electron transport in many metabolic reactions and also their ability to be reduced by the incidence of light. Lisa Wulund 2015. When activated by light, cryptochromes undergo a redox cycle, in the course of which radical pairs are generated during photo-reduction as well as during light-independent re-oxidation.
  • In plants, iron uptake and homeostasis are critical for survival, and these processes are tightly regulated at the transcriptional and posttranscriptional levels. circadian clock controls many physiological processes through rhythmic expression of transcripts. The expression of three Fe homeostasis genes (IRON REGULATED TRANSPORTER1 [IRT1], BASIC HELIX LOOP HELIX39, and FERRITIN1) in Arabidopsis (Arabidopsis thaliana) (using promoter:LUCIFERASE transgenic lines) showed circadian regulation of transcription. The circadian clock monitors a number of clock outputs and uses these outputs as inputs to modulate clock function.  Iron deficiency results in a lengthened circadian period. S Hong 2013.
  • Haem has been previously implicated in the function of the circadian clock, but whether iron homeostasis is integrated with circadian rhythms was unknown. However RNA interference (RNAi) screen using clock neurons of Drosophila melanogaster has been described. RNAi is targeted to iron metabolism genes, including those involved in haem biosynthesis and degradation. The results indicate that Ferritin 2 Light Chain Homologue (Fer2LCH) is required for the circadian activity of flies kept in constant darkness. Oscillations of the core components in the molecular clock, PER and TIM, were also disrupted following Fer2LCH silencing. Other genes with a putative function in circadian biology include Transferrin-3, CG1358 (which has homology to the FLVCR haem export protein) and five genes implicated in iron-sulfur cluster biosynthesis: the Drosophila homologues of IscS (CG12264), IscU (CG9836), IscA1 (CG8198), Iba57 (CG8043) and Nubp2 (CG4858). Therefore, Drosophila genes involved in iron metabolism are required for a functional biological clock. K Mandilaras 2012. There is also reciprocal regulation of haem biosynthesis and the circadian clock in mammals. K Kaasik 2004.


This article merely joins up other peoples work into an overall system.  These works have been referenced so it is clear that others have provided the individual pieces of evidence that have been used to shape a specific systems approach.    




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