1

1. Heme proteins in general

2

3 3

Cloning, expression and purification

E. coli K-12 genome

cloning

pET21c

overexpression Full-length YddV

97

66

45

(kDa)

54 kDa purification

YddV contains a b-type heme with 1 : 1 stoichiometry.

33

Myoglobin O2 Storage

Myoglobin, Hemoglobin: Heme iron complex containing proteins

(Haemoglobin, Haem: UK spelling) 4

5

You don’t need O2. Less myoglobin.

You need O2 because of continuous swimming. More myoglobin.

1. Heme Proteins (Haem Proteins: UK)

Heme iron complex = Heme (Haem: UK)

Iron Fe(II), Fe2+, Ferrous

Fe(III), Fe3+, Ferric

+

Heme Porphyrin, Protoporphyrin IX

Hemin = Protoporphyrin IX-Fe(III) complex

2. Non-heme Iron Proteins

6

Figure 2. Chemical structures of representative iron complexes in proteins. Rubredoxin, ferredoxin, [4Fe–4S] clusters, and Rieske [2Fe–2S] clusters are found in proteins that function as electron transfer processes. [2Fe–2S] and [4Fe–4S] clusters are also engaged in regulation of the transcription of genes that are under redox control. Mononuclear iron complexes are found in enzymes that catalyze oxygenation reactions. Oxo-Fe2 complexes catalyze dehydrogenation or reduction of ribonucleotides to deoxyribonucleotides. Fe–Ni hydrogenase and Fe–Fe hydrogenase complexes are found in hydrogenases. The CO dehydrogenase cluster shown is cluster B in carbon monoxide dehydrogenase. The aconitase intermediate occurs as the dehydration intermediate in aconitase. Iron protoporphyin IX is found in heme proteins, cytochromes c and a, and cytochrome P450. The radical SAM initiation complex is found in radical SAM enzymes. FeMoco and P-cluster are essential cofactors in nitrogenase. The interstitial carbide (C4–) has recently been identified.(14, 15) ACS Chem. Biol. 7, 1477 (2012) Radical SAM enzymes use a 4Fe-4S cluster to transfer an electron from an external source

(such as flavodoxin) to an S-adenosylmethionine (SAM) molecule, which converts to methionine and a 5’-deoxyadenosyl radical. This radical is very reactive, and is able to abstract a proton from a C-H that is appropriately placed.

7

Iron Protoporphyrin IX, heme b Prosthetic group A flat and planar structure. Itself Toxic O2

-., H2O2 are formed.

Heme a Heme a is a form of heme found In cytochromes a and a3.

8

9

Heme proteins = heme + proteins

Transfer & storage of gas molecules

O2, NO

Mb, Hb

Oxidation

O2 + e-

P450, NOS

Electron transfer

e-

Cytochrome c

Heme sensor Gas sensor

Heme sensor: HRI Gas (O2 , CO, NO) sensor: FixL, DOS

10

11

Emergent roles of heme in signal transduction: Heme sensors and gas sensors 1. Heme proteins in general 1-1. Iron and heme: Metabolism and hemeostasis 1-2. Myoglobin and hemoglobin: Oxygen binding and new functions 1-3. Cytochrome c and cytochrome b5: Electron transfer and new functions 1-4. Cytochrome P450: Oxygen activation and biodiversity 1-5. Nitric oxide synthase: Oxygen activation and NO-related functions 1-6. Heme oxygenase: Oxygen activation and CO-related functions 2. Heme sensors 2-1. General concepts and examples 2-2. Heme-regulated eukaryotic initiation factor 2a (eIF2a) kinase 2-3. Heme sensors associated with circadian rhythms and transcription

12

Emergent roles of heme in signal transduction: Heme sensors and gas sensors 3. Heme-based gas sensors 3-1. Oxygen sensors: FixL, EcDOS, YddV, AfGcHK 3-2. NO sensors: soluble guanylate cyclase (sGC), HNOX 3-3. CO sensors: CooA, cystathionine b–synthase (CBS), BK channel 3-4. H2S: Synthesis and functions 4. Relationships between O2, NO, CO and H2S

5. Reactive oxygen species (ROS)

6. Non-heme oxygen sensor: Hypoxia-inducible factors (HIF1a)

7. Seminars

Cytochrome c plays a key part in electron transport associated with aerobic cellular respiration. Cytochrome c is a small heme protein which is associated with the inner membrane of the mitochondria. In the electron transport process it transfers electrons between Complex III and Complex IV.

13

14

NSAID: non-steroid anti-inflammatory drug

Syntheses of steroids such as sex hormones

15

Blue Rose: Suntory

Flower color was changed by manipulation of P450 genes: Rose

Heme-assisted S-nitrosation of a proximal thiolate in

a nitric oxide transport protein

Proc. Natl. Acad. Sci. USA 102, 594 (2005)

16 Rhodnius prolixus (the kissing bug)

Cimex lectularius (the bedbug)

Vasorelaxation +

Platelet Inhibition

17

Vasorelaxation

Platelet Inhibition

Soluble guanylate

cyclase (sGC)

sGC, eNOS: Heme protains

Figure 12. Summarized illustration of the main, significant categories of NO reactions in the vascular system. The reactions include (1) reactions with metal centers (mainly heme); (2) nitration (protein tyrosine); (3) S-nitrosylation, or the interaction of NO with cysteine sulfahydryls/thiol, where a nitrosyl group is added post-translationally; (4) free-radical interactions; (5) reactions with plasma O2; and (6) synthesis of cGMP through the catalysis of sGC by NO, then leading to the activation of protein kinases and phosphodiesterases.

18

19

1-1. Iron and heme: Metabolism and homeostasis

Mechanisms of Mammalian Iron Homeostasis

Biochemistry 51, 5705 (2012)

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors

Arch. Biochem. Biophys. 592, 60 (2016)

The Iron age of host-microbe interactions

EMBO Reports 16, 1482 (2015)

Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death, and Disease

Trends in Biochemical Sciences 41, 274 (2016)

Heme: Modulator of Plasma Systems in Hemolytic Diseases Trends Mol. Med. 22, 200 (2016)

Iron metabolism: a double-edged sword in the resistance of glioblastoma to therapies

Trends Endocrinol. Metab. 26, 322 (2015)

Ironing out Ferroportin

Cell Metab. 22, 1 (2015)

The importance of eukaryotic ferritins in iron handling and cytoprotection

Biochem. J. 472, 1 (2015)

Iron-associated biology of Trypanosoma brucei

Biochim. Biophys. Acta 1860, 363 (2016)

20

Mechanisms of Mammalian Iron Homeostasis Biochemistry 51, 5705 (2012) ABSTRACT: Iron is vital for almost all organisms because of its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for oxygen and energy metabolism, as well as for several other essential processes. Mammalian cells utilize multiple mechanisms to acquire iron. Disruption of iron homeostasis is associated with various human diseases: iron deficiency resulting from defects in the acquisition or distribution of the metal causes anemia, whereas iron surfeit resulting from excessive iron absorption or defective utilization causes abnormal tissue iron deposition, leading to oxidative damage. Mammals utilize distinct mechanismsto regulate iron homeostasis at the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance. This review outlines recent advances in iron regulatory pathways as well as in mechanisms underlying intracellular iron trafficking, an important but less studied area of mammalian iron homeostasis.

21

22

23

24

Mammalian Mechanisms of Iron Homeostasis

Biochemistry 51, 5705 (2012)

Fig. 1. Iron absorption, distribution, and recycling in

the body and quantitative exchange of iron between

body iron sources. Body iron levels are maintained by

daily absorption of ~1-2 mg of dietar iron to account

for obligatory losses of a similar amount of iron

through sloughing of mucosal and skin cells,

hemorrhage, and other losses. Approximately 4 mg of

iron is found in circulation bound to Tf, which

accounts for 0.1% of the total body iron. Majority of

the body iron is found in the erythroid compartment of

bone marrow and in mature erythrocytes contained

within the heme moiety of the

hemoglobin. Splenic reticuloendothelial macrophages,

which recycle iron from senescent red blood cells,

provide iron for the new red blood cell synthesis. Tf

delivers iron to developing erythroid precursors, as

well as to other sites of iron utilization. Liver

hepatocytes store iron in ferritin shells. During

pregnancy, 250 mg of iron is transported across the

placenta to the fetus. The distribution of iron in the

body is altered in iron deficiency and iron overload

(see text).

25

Fig. 2. Regulation of systemic iron

metabolism. Organs and cell types

involved in systemic iron balance are

shown. Duodenal enterocytes absorb

dietary iron via DMT1 located on the

apical surface upon reduction of Fe3+ to

Fe2+ by DcytB. Spleenic

reticuloendothelial macrophages recycle

iron from senescent red blood cells.

Both cell types release iron via

ferroportin with the aid of hephaestin,

which oxidizes Fe2+ to Fe3+. Iron is

also oxidized by ceruloplasmin in the

circulation. Plasma Tf captures and

circulates iron in the body. Hepatic

hormone, hepcidin regulates iron efflux

from these cells by regulating the

stability of ferroportin. Synthesis and

secretion of hepcidin by hepatocytes is

influenced

by iron levels in the body as well as

conditions that affect iron metabolism

indirectly such as

inflammation, ER stress, erythropoiesis,

and hypoxia (see text for additional

details).

26

Fig. 3. Cellular iron metabolism. Most

cells in the body obtain iron from

circulating differic Tf. Iron loaded holo-

Tf binds to TfR1 on the cell surface and

the complex undergoes endocytosis via

clathrin coated pits. A proton pump

acidifies the endosome resulting in the

release of Fe3+, which is subsequently

reduced to Fe2+ by Steap3 and

transported across the endosomal

membrane to the cytosol by DMT1.

DMT1 also facilitates dietary iron

absorption. Apo-Tf is recycled back to the

cell surface and released from TfR1 to

plasma to repeat another cycle. Newly

acquired iron enters into cytosolic “labile

iron pool” (LIP), which is redox-active.

LIP is chelated by intracellular

siderophore that facilitates intracellular

iron trafficking to mitochondria via an

unknown receptor for metabolic

utilization (such as synthesis of heme and

iron-sulfur clusters), and cellular iron that

is not utilized is either stored in ferritin or

exported via ferroportin. Cells also export

iron contained in ferritin and heme.

27

Fig. 4. Cellular iron balance. A typical IRE motif consists of a hexanucleotide loop with the sequence 5′-CAGUGH-3′ (H

could be A, C, or U) and a stem, interrupted by a bulge with an unpaired C residue. IREs post-transcriptional control

expression of regulators of cellular iron metabolism in concert with IRPs. Translational-type IRE/IRP interactions in the 5’

UTR modulate the expression of the mRNAs encoding H- and L-ferritin, ALAS2, m-aconitase, ferroportin, and HIF-2α,

which in turn control iron storage, erythroid iron utilization, energy homeostasis, iron efflux, and hypoxic responses,

respectively. Conversely, IRE/IRP interactions in the 3’ UTR stabilize the mRNAs encoding TfR1, DMT1, and Cdc14A,

which are involved in iron uptake, iron transport, and the cell cycle, respectively. Under physiological conditions, IRP1 is

regulated by a reversible ISC switch. Iron deficiency, promotes ISC disassembly and a conformational rearrangement,

resulting in conversion of IRP1 from c-aconitase to an IRE-binding protein. The ISC is regenerated in iron-replete cells.

Hypoxia favors maintenance of the ISC, while H2O2 promotes its disassembly. When the ISC biogenesis pathway is not

operational, iron leads to ubiquitination of apo-IRP1 by the FBXL5 E3 ligase complex (including Skp1, Cul1 and Rbx1),

resulting in proteasomal degradation. IRP2 is stable in iron deficient and/or hypoxic cells; under these conditions FBXL5

undergoes ubiquitination and proteasomal degradation. An increase in iron and oxygen levels stabilizes FBXL5 by

formation of an Fe-O-Fe center in its hemerythrin domain, triggering the assembly of an E3 ubiquitin ligase complex

together with Skp1, Cul1 and Rbx1. This complex ubiquitinates IRP2, leading to its recognition by the proteasome and its

degradation.

28

Supplementation With Oral vs. Intravenous Iron for Anemia With IBD (inflammatory bowel disease) or Gastrointestinal Bleeding: Is Oral Iron Getting a Bad Rap? Amer. J. Gastroenterology 106, 1872 (2011) Ferric iron (Fe3+) is reduced to ferrous iron (Fe2+) in the intestinal lumen by ferrireductase. Ferrous iron (Fe2+) is transported across the enterocyte by divalent metal ion transporter-1 (DMT-1). Dietary heme iron is transported across the enterocyte by heme carrier protein 1 (HCP1). Inside the enterocyte, heme is acted upon by heme oxygenase, which releases ferrous iron from the protoporphyrin in heme. The released iron enters the same pool as dietary non-heme iron. A portion of this pool is stored as ferritin inside the enterocyte, which is later lost with sloughing of the intestinal mucosa. The remainder of the ferrous iron is transported across the basolateral membrane of the enterocyte by ferroportin. The transported Fe2+ is oxidized back to the Fe3+ form by ferroxidase. Fe3+ is then incorporated into transferrin in the serum. The protein hepcidin regulates iron transport into serum by causing internalization of ferroportin with subsequent lysosomal breakdown inside the enterocyte.

29

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors Arch. Biochem. Biophys. 592, 60 (2016) ABSTRACT In humans, the bulk of iron in the body (over 75%) is directed towards heme- or FeeS cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and FeeS cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways

30

31

32

33

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The Iron age of host–microbe interactions EMBO Reports 16, 1482 (2015) Abstract Microbes exert a major impact on human health and disease by either promoting or disrupting homeostasis, in the latter instance leading to the development of infectious diseases. Such disparate outcomes are driven by the ever-evolving genetic diversity of microbes and the countervailing host responses that minimize their pathogenic impact. Host defense strategies that limit microbial pathogenicity include resistance mechanisms that exert a negative impact on microbes, and disease tolerance mechanisms that sustain host homeostasis without interfering directly with microbes. While genetically distinct, these host defense strategies are functionally integrated, via mechanisms that remain incompletely defined. Here, we explore the general principles via which host adaptive responses regulating iron (Fe) metabolism impact on resistance and disease tolerance to infection.

35

Miguel P Soares, and Günter Weiss EMBO Rep. 2015;16:1482-1500

Interrelationship of Fe and Heme metabolism More than 80% of the bioavailable

Fe in mammals exists in the form of heme contained in hemoproteins [241].

36

Miguel P Soares, and Günter Weiss EMBO Rep. 2015;16:1482-1500

Regulation of Fe metabolism in response to extracellular pathogens (A) Immune responses

to extracellular pathogens encompass the production of cytokines, for example, IL‐1, IL‐6, and IL‐22, which induce the transcription of the hepcidin (HAMP) gene in hepat..

37

Miguel P Soares, and Günter Weiss EMBO Rep. 2015;16:1482-1500

Regulation of Fe metabolism in response to intracellular pathogens Mø

activation in response to intracellular pathogens is associated with a reduction

of intracellular Fe levels.

38

Miguel P Soares, and Günter Weiss EMBO Rep. 2015;16:1482-1500

Fe/heme metabolism and disease tolerance to infection RBC are present at

high numbers throughout the body [49].

39

Copyright © 2015 Elsevier Ltd Terms and Conditions

Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death,

and Disease

Trends in Biochemical Sciences 41, 274 (2016)

Iron is necessary for life,but can also cause cell death. Accordingly, cells

evolved a robust, tightly regulated suite of genes formaintaining iron

homeo-stasis. Previous mechanistic studies on iron homeostasis have

granted insight into the role of iron in human health and disease. We

highlight new regulators of iron metabolism, including iron-trafficking

proteins [solute carrier family39, SLC39,also known as ZRT/IRT-like protein,

ZIP; and poly-(rC)-binding protein, PCBP] and acargo receptor (NCOA4)

that is crucial for release of ferritin-bound iron. We also discuss emerging

roles of iron in apoptosis and anovel iron-dependent cell death pathway

termed ‘ferroptosis’, the dysregulation of iron metabolism in human

pathologies, and the use of iron chelators in cancer therapy.

Figure 1 Trends in Biochemical Sciences DOI: (10.1016/j.tibs.2015.11.012)

Copyright © 2015 Elsevier Ltd Terms and Conditions

41

Figure 2 Trends in Biochemical Sciences DOI: (10.1016/j.tibs.2015.11.012) Copyright © 2015 Elsevier Ltd Terms and Conditions 42

Figure 3

Trends in Biochemical Sciences DOI: (10.1016/j.tibs.2015.11.012)

Copyright © 2015 Elsevier Ltd Terms and Conditions

43

Figure 4 Trends in Biochemical Sciences DOI: (10.1016/j.tibs.2015.11.012)

Copyright © 2015 Elsevier Ltd Terms and Conditions

44

Box I

Trends in Biochemical Sciences DOI: (10.1016/j.tibs.2015.11.012)

Copyright © 2015 Elsevier Ltd Terms and Conditions

45

Respiratory syncytial virus (RSV) infection enhances Pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity Proc. Nat. Acad. Sci. USA 113, 1642 (2016) 46

47

Respiratory disorders: Ironing out smoking-related airway disease Nature March 9, 2016 doi:10.1038/nature17309 Nat. Med. 22, 163 (2016) Figure 1: Regulation of mucociliary clearance in chronic obstructive pulmonary disease (COPD). Smoking increases the risk of COPD, during which airways in the lungs become obstructed by mucus. Cloonan et al report that expression of the iron regulatory protein IRP2 contributes to this process. Specifically, their data indicate that exposure to smoke leads to increased IRP2 levels, which promotes iron accumulation in mitochondria. This increased iron loading in turn increases the activity of the protein cytochrome c oxidase (COX), and, together, these factors lead to mitochondrial dysfunction in lung cells. Compromised mitochondrial function reduces the ability of cellular protrusions called cilia to perform their normal role in clearing mucus. The drug deferiprone, which blocks the toxic effects of excess iron in mitochondria, preserves ciliary function in smoke-exposed mice.