INTRODUCTIONThe physiological function of haemoglobinsin species other than vertebrate animals has not been well established.Althoughthe role of vertebrate haemoglobins as facilitators of oxygen diffusion is wellestablished, the function of globins in invertebrate animals, as well as inplants, protozoa, fungi, and bacteria, is generally unclear. The presentdata suggest that, despite the high degree of homology between globins ofprokaryotic and eukaryotic microorganisms, each species may have developed adiscrete role for its hemoglobin.

1 There has been extensive comparative research of thehaemoglobins of micro-organisms-yeast, bacteria, invertebrates/vertebrates(mammals), and humans; to identify the presence similar parallel oxygen-sensingsystems in all organisms and understand the functions of these highly conservedhaemoglobin proteins. The aerobic bacteria, Vitreoscilla single-domain haemoglobin(VtHb) has been extensively studied in this regard.Whereas most vertebrate haemoglobinsare composed of two types of polypeptide subunits, ? and ?, which have singleheme domains and form ?2?2 tetramers; invertebrate and microbial haemoglobinsare more varied.

Many of the bacterial and fungal haemoglobins that have beencharacterized to date fall into two general categories: dimeric hemoproteinscomposed of two single heme domain polypeptides and monomeric flavohaemoproteinscomposed of a single polypeptide containing a single heme-binding domain and asingle Flavin-binding domain.2 These flavohaemoglobins(flavoHbs) are made of a globin domain fused with a ferredoxin reductase-likeFAD- and NAD-binding modules. These proteins are widely represented amongbacteria and yeasts3   GENEREGULATIONHaemoglobins recently sequencedfrom the bacteria E. coli and Alcaligenes eutrophus and the yeasts Saccharomyces cerevisiae (baker’syeast) and Candida norvegensis are two-domain proteins with nearly 40% sequenceidentity.

The flavohaemoproteins contain bothheme and flavin binding domains and which are capable of transferring electronsfrom NADPH to heme iron.2  Their N-terminal regions sharesubstantial sequence homology with the single-domain of the aerobic bacteriaVitreoscilla (VtHb), whereas the C terminus contains a reductase domain withpotential binding sites for flavin (FAD) and NADPH. 4  Baker’s yeast is a facultativeaerobe, capable of growth in the complete absence of molecular oxygen, conditionsunder which yeast cells can grow but under which most mammalian cells, letalone the intact organism, could not long survive.

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5 The S. cerevisiae flavohaemoglobingene (YHB) has been mapped to chromosome 7, near the formyltetrahydrofolatesynthase (ADE3) locus. The genes involved in S.

cerevisae oxygen regulation aredivided into two broad categories- (1) genes regulated by heme and (2) geneswhose regulation is heme independent. Heme-regulated genes fall into twoclasses: heme-activated and heme-repressed genes. Activation of theflavohaemoglobin gene is achieved through one of two transcriptionalactivators, the heme-dependent HAP1 protein or the heme-activated,glucose-repressed HAP2/3/4 complex.6 S. cerevisiae globin message isinduced during logarithmic growth and under oxygen-replete conditions. YHB1 isexpressed in hyperoxic states and conditions that promote oxidative stress.These findings suggest that YHb plays a role in the oxidative stressresponse in yeast functioning as an oxygen scavenger. 2 YHb is not essential for cellgrowth.

Normally, actively respiring yeast cells have very low levels of the flavohaemoglobinand flavohaemoglobin gene disruption does not alter cell viability or growth ina variety of oxygen conditions and carbon sources. This protein binds oxygenreversibly only when NADPH is present, indicating that it has an NAD(P)Hreductase activity for the heme domain. However, when the mitochondrialelectron transport chain has been compromised by either mutation (i.e. thedeletion of the mitochondrial genome) or respiration inhibitors (e.g. antimycinA)- the expression of the flavohemoglobin gene, YHB1 is upregulated.2 Saccharomyces cerevisiae alsoexpresses several isozymes of cytochrome c oxidase.

Isozymeswhich incorporate the Vb isoform have both higher turnover rates and higherrates of heme oxidation than isozymes which incorporate Va.YHB1 level has been shown to increase in cells engineered to express thehypoxic isoform-Vb, of cytochrome c oxidase subunit V under aerobic conditions.Regulation of the S.

cerevisiaeflavohemoglobin gene is clearly controlled by cell density and oxygen tensionbut in a manner different from that of the bacterial globins previouslystudied. There is Repression of YHG mRNA with increasing cell density.The repression of endogenous YHGgene expression upon exit from exponential phase may be due to a combination offactors such as hypoxic conditions, induction of a repressor, or the alteredchromatin structure at higher culture densities that has been implicated inrepressing most exponential phase mRNA species In S. cerevisiae there are twoclasses of yeast genes: “aerobic” genes (e.g.

HMG1, COX5a, CYC1, AAC1, AAC2,TIF51a) , which are expressed optimally in the presence of air, and “hypoxic”genes (e.g. HEM13, ERG11, HMG2, CPR1, SUT1, OLE1, COX5b, CYC7, AAE3, ANB1,TIF51b), which are expressed optimally at low oxygen concentrations2 S. cerevisae flavohaemoglobin (YHG)transcriptional activation is modulated by oxygen concentration.

Below 0.1%oxygen concentration aerobic activation of the YHG promoter ends and theanaerobic expression of the YHG message begins. Transcript levels for YHB1 arereduced in the absence of oxygen (lane 1) and slightly elevated under hyperoxicconditions (lane 3).

Thus, 0.1% likely straddles the concentration of oxygennecessary for heme biosynthesis.                                                            Increasingoxygen tensionOxygen availability is sensed throughcellular heme levels. Consequently, under hypoxic conditions, heme levels arereduced. 5  In S. cerevisiae, transcription factorsinvolved in oxygen regulation of gene expression have been well characterized. Aerobicexpression of YHG is predominantly activated by the HAP1 and HAP2/3/4transcription factor complexes. An anaerobic system to produce YHG messageindependent of the HAPs is also present.

Although there is a decrease in YHGgene expression as the cells approach anaerobiosis. Hap1 is aprotein composed of a zinc finger DNA binding domain at the N-terminus, a hemebinding domain within the central region, and a transcriptional activationdomain at the C-terminus.5 YHGpromoter/lacZ fusion construct is also regulated by heme and the HAPsTIF51A/B gene pair also regulateheme biosynthesis. Under aerobic conditions, heme accumulates and serves as aneffector for the transcriptional activator Hap1.

The heme-Hap1 complexactivates transcription of the ROXI gene that encodes a repressor of one set ofhypoxic genes. For example, above 0.1% Oxygen concentration, only TIF51A ispresent; below this concentration, only TIF51B can be detectedYHG is probably not involved infacilitating oxygen storage or diffusion during hypoxic electron transport.Contrary to many other genes involved in respiration, the YHG messagerepresents the first known example of a HAP2/3/4-regulated gene that is notglucose-repressed, indicating that the gene may be required in both fermentableand non-fermentable carbon sources. There is an increase in YHG mRNA levels incells grown under high oxygen tension. This phenomenon can be attributed toincreased heme levels, which also stimulate superoxide dismutase and catalasetranscription in S. cerevisiae.

Therefore, flavohemoglobin in S. cerevisiae mayalso be involved in the detoxification of oxygen.Cells sense hypoxia through theinability to maintain oxygen-dependent heme biosynthesis.

Under hypoxicconditions, heme levels fall, and a heme-deficient Hap1 complex represses ROX1expression. When hemeis bound, Hap1 acts as a transcriptional activator of genes containing itsrecognition site (5’CGGN6CGG) -for the most part, genes encoding a variety ofrespiratory and oxidative stress functions i.e. aerobic genes. In addition, the ROX1 gene encoding the repressor of hypoxicgenes is also activated by the Hap1-heme complex. The Rox1 repressor binds toits cognate site upstream of the hypoxic genes to repress their transcription.Under hypoxic or anaerobic growth conditions, heme levels are reduced. Hap1still binds to its cognate site, but in the absence of heme, additionalproteins bind to Hap1 creating a larger complex that represses transcription.

HAP1directly represses its own transcription by binding HAP1 promoters. Consequently,under hypoxic conditions, ROX1 expression is repressed resulting in thederepression of the hypoxic genes. Therefore, heme is the effector molecule.  Rox1 consists of 368 amino acids that can be divided intothree domains.

The first third of the protein consists of an HMG DNA bindingmotif. This motif is found in a number of DNA binding and bending proteinsincluding both sequence-specific regulatory proteins and general DNA bindingchromatin proteins. The ability of these proteins to bend DNA has led to theirdesignation as architectural proteins, proteins that induce conformations inDNA that may be important for their function. Using gel retardation and DNase protection studies, thisdomain was found to bind as a monomer to the previously mapped operator sitesin the ANB1 gene. Experiments with chimeric proteins suggests that this bend isnot essential but enhances repression activity.The Rox1 protein consists of at least two additionaldomains. There is a glutamine-rich region following the HMG domain, extendingfrom residues 100 to 123.

Deletion of this region does not affect either the functionor the lability of Rox1, and its purpose is not known. The remainder of theprotein represents the repression domain. Deletion of this domain results in aDNA binding protein with no repression activity, and fusion of this domain to aheterologous DNA binding protein (the amino terminus of the yeast protein Ga14)results in a chimeric protein with repressor activity.The binding of transcriptional activators often lieshundreds of base pairs from the TATA box, where the basal transcriptionalmachinery and RNA polymerase II bind. Many genes have multiple, differentiallyregulated activators that can act individually and additively to settranscription levels. Such promoters preclude repression mechanisms that simplyact through competitive binding of the repressor with an activator or RNApolymerase, and suggest that repression must be an active process. Rox1 binding to DNA alone is notsufficient to repress hypoxic gene transcription.

A general repression complex consistingof two proteins, Tup1 and Ssn6, is also required which functions via the HOG-MAPkinase signalling pathway.  CONCLUSIONAs in all organisms, geneexpression in S. cerevisae is a complex process. Despite the highly conserved natureof the globin gene among many species including man, and the extensivesequence homology between baker’s yeast and Candida as well as those of severalbacterial species, theS. cerevisiae flavohemoglobin gene (YHB1) may have distinct transcriptionalactivation mechanisms with consequent unique function for its flavohemoprotein(YHb). 1 S.

cerevisiae YHb is an importantregulator of many other genes, regulating the expression and transcription ofseveral genes based on oxygen concentration in hypoxic or aerobic environments.The  S. cerevisiae heme activator proteinHap1 binds to DNA and activates transcription of genes encoding functions requiredfor respiration and for controlling oxidative damage, in response to heme.7     BIBLIOGRAPHY1.        Crawford MJ,Sherman DR, Goldberg DE. Regulation of Saccharomyces cerevisiae flavohemoglobingene expression.

J Biol Chem Internet. 1995 Mar 24;270(12):6991–6. 2.        Zhao XJ, RaittD, V Burke P, Clewell AS, Kwast KE, Poyton RO. Function and expression of flavohemoglobinin Saccharomyces cerevisiae. Evidence for a role in the oxidative stressresponse. J Biol Chem Internet.

1996 Oct 11;271(41):25131–8. 3.        Bonamore A,Boffi A. Flavohemoglobin: Structure and reactivity. IUBMB Life Internet. 2007Dec 12;60(1):19–28. 4.        Zhu H, RiggsAF.

Yeast flavohemoglobin is an ancient protein related to globins and areductase family. Biochemistry Internet. 1992;89:5015–9. 5.        Zitomer RS,Carrico P, Deckert J. Regulation of hypoxic gene expression in yeast.

KidneyInt Internet. 1997;51(2):507–13. 6.        Zitomer RS,Lowry C V.

Regulation of gene expression by oxygen in Saccharomyces cerevisiae.Microbiol Rev Internet. 1992 Mar 1;56(1):1–11. 7.

       Hon T, Lee HC,Hu Z, Iyer VR, Zhang L. The Heme Activator Protein Hap1 Represses Transcriptionby a Heme-Independent Mechanism in Saccharomyces cerevisiae.